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TRANSLATIONAL MODELS OF CORONARY ARTERY DISEASE, MYOCARDIAL INFARCTION AND HEART FAILURE Development, Validation and in vivo Imaging Studies Using Positron Emission Tomography Miikka Tarkia TURUN YLIOPISTON JULKAISUJA – ANNALES UNIVERSITATIS TURKUENSIS Sarja - ser. D osa - tom. 1175 | Medica - Odontologica | Turku 2015 University of Turku Faculty of Medicine Institute of Clinical Medicine Department of Clinical Physiology and Nuclear Medicine and Turku PET Centre Drug Research Doctoral Programme at University of Turku Supervised by Professor Anne Roivainen, PhD Turku PET Centre and Turku Centre for Disease Modelling University of Turku Turku, Finland Associate Professor Antti Saraste, MD, PhD Turku PET Centre and Heart Center University of Turku and Turku University Hospital Turku, Finland Reviewed by Adjunct Professor Esko Kankuri, MD, PhD Department of Pharmacology University of Helsinki Helsinki, Finland Adjunct Professor Kirsi Timonen, MD, PhD Department of Clinical Physiology and Nuclear Medicine Central Hospital of Central Finland and University of Eastern Finland Kuopio, Finland Opponent Professor Johanna Kuusisto, MD, PhD Department of Medicine University of Eastern Finland Kuopio, Finland The originality of this thesis has been checked in accordance with the University of Turku quality assurance system using the Turnitin OriginalityCheck service. ISBN 978-951-29-6129-0 (PRINT) ISBN 978-951-29-6130-6 (PDF) ISSN 0355-9483 Painosalama Oy - Turku, Finland 2015 To Anna and Maria 4 Abstract ABSTRACT Miikka Tarkia TRANSLATIONAL MODELS OF CORONARY ARTERY DISEASE, MYOCARDIAL INFARCTION AND HEART FAILURE Development, validation and in vivo imaging studies using positron emission tomography Department of Clinical Physiology and Nuclear Medicine and Turku PET Centre, University of Turku, Turku, Finland Coronary artery disease is an atherosclerotic disease, which leads to narrowing of coronary arteries, deteriorated myocardial blood flow and myocardial ischaemia. In acute myocardial infarction, a prolonged period of myocardial ischaemia leads to myocardial necrosis. Necrotic myocardium is replaced with scar tissue. Myocardial infarction results in various changes in cardiac structure and function over time that results in “adverse remodelling”. This remodelling may result in a progressive worsening of cardiac function and development of chronic heart failure. In this thesis, we developed and validated three different large animal models of coronary artery disease, myocardial ischaemia and infarction for translational studies. In the first study the coronary artery disease model had both induced diabetes and hypercholesterolemia. In the second study myocardial ischaemia and infarction were caused by a surgical method and in the third study by catheterisation. For model characterisation, we used non-invasive positron emission tomography (PET) methods for measurement of myocardial perfusion, oxidative metabolism and glucose utilisation. Additionally, cardiac function was measured by echocardiography and computed tomography. To study the metabolic changes that occur during atherosclerosis, a hypercholesterolemic and diabetic model was used with [18F] fluorodeoxyglucose ([18F]FDG) PET-imaging technology. Coronary occlusion models were used to evaluate metabolic and structural changes in the heart and the cardioprotective effects of levosimendan during post-infarction cardiac remodelling. Large animal models were used in testing of novel radiopharmaceuticals for myocardial perfusion imaging. In the coronary artery disease model, we observed atherosclerotic lesions that were associated with focally increased [18F]FDG uptake. In heart failure models, chronic myocardial infarction led to the worsening of systolic function, cardiac remodelling and decreased efficiency of cardiac pumping function. Levosimendan therapy reduced post-infarction myocardial infarct size and improved cardiac function. The novel 68Ga-labeled radiopharmaceuticals tested in this study were not successful for the determination of myocardial blood flow. In conclusion, diabetes and hypercholesterolemia lead to the development of early phase atherosclerotic lesions. Coronary artery occlusion produced considerable myocardial ischaemia and later infarction following myocardial remodelling. The experimental models evaluated in these studies will enable further studies concerning disease mechanisms, new radiopharmaceuticals and interventions in coronary artery disease and heart failure. Keywords: coronary artery disease, myocardial ischaemia, myocardial infarction, heart failure, positron emission tomography (PET) Tiivistelmä5 TIIVISTELMÄ Miikka Tarkia SEPELVALTIMOTAUDIN, SYDÄNINFARKTIN JA SYDÄMEN VAJAATOIMINNAN TRANSLATIONAALISET MALLIT Kehitys, validointi ja in vivo kuvantaminen positroniemissiotomografialla Kliininen fysiologia ja isotooppilääketiede ja Valtakunnallinen PET-keskus, Turun yliopisto, Turku, Suomi Sepelvaltimotaudissa sepelvaltimot ahtautuvat ateroskleroosin vuoksi, mikä johtaa sydänlihaksen verenvirtauksen heikkenemiseen ja sydänlihasiskemiaan. Akuutissa sydäninfarktissa pitkittynyt iskemia johtaa sydänlihaskuolioon eli nekroosiin, joka korvautuu arpikudoksella. Sydäninfarktin seurauksena sydämen rakenteessa ja toiminnassa tapahtuu muutoksia, mikä johtaa niin kutsuttuun haitalliseen sydänlihaksen uudelleenmuovautumiseen ja lopulta sydämen toiminnan heikkenemiseen ja kroonisen vajaatoiminnan kehittymiseen. Tässä väitöskirjatyössä kehitettiin ja validoitiin kolme erilaista sepelvaltimotaudin, sydänlihasiskemian ja infarktin suureläinmallia translationaalisia tutkimuksia varten. Ensimmäisessä osatyössä sepelvaltimotaudin mallissa oli yhdistetty diabetes ja hyperkolesterolemia. Toisessa osatyössä sydänlihasiskemia ja infarkti oli aiheutettu kirurgisesti ja kolmannessa osatyössä katetrisaatiolla. Mallien karakterisoinnissa käytettiin kajoamattomia positroni emissiotomografiaan (PET) perustuvia kuvantamismenetelmiä, joilla mitattiin sydänlihaksen verenvirtausta, hapenkulutusta ja glukoosinkäyttöä. Lisäksi sydämen pumppausominaisuuksia mitattiin ultraäänikardiografialla ja tietokonetomografialla. Hyperkolesterolemia ja diabetesmallilla tutkittiin ateroskleroottisten tautimuutosten kuvantamista [18F]fluorodeoksi glukoosi ([18F]FDG) PET-merkkiaineella. Sepelvaltimon ahtauman malleilla tutkittiin sydämen metabolisia ja rakenteellisia muutoksia ja levosimendaanin sydäntä suojaavaa vaikutusta. Tutkimme myös uusia radiolääkeaineita sydänlihaksen verenvirtauksen määrittämiseen. Sepelvaltimotaudin mallissa havaitsimme ateroskleroottisia muutoksia, jotka olivat yhteydessä lisääntyneeseen [18F]FDG kertymään. Sydämen vajaatoiminnan malleissa krooninen sydäninfarkti johti systolisen funktion huononemiseen, haitalliseen sydänlihaksen uudelleenmuovautumiseen ja alentuneeseen sydänlihaksen hyötysuhteeseen. Infarktinjälkeinen hoito levosimendaanilla johti infarktialueen rajoittumiseen ja sydämen pumppausominaisuuksien parantumiseen. Uudet testatut 68Ga-leimatut radiolääkeaineet eivät tässä tutkimuksessa soveltuneet sydänlihaksen verenvirtauksen määrittämiseen. Johtopäätöksenä, diabetes ja hyperkolesterolemia johtavat varhaisen vaiheen ateroskleroottisiin muutoksiin. Sepelvaltimon tukkeuma kehitti merkittävän sydänlihasiskemian ja myöhemmän infarktin johtaen sydänlihaksen uudelleenmuovautumiseen. Testattujen koemallien myötä meillä on mahdollisuus tutkia lisää sepelvaltimotaudin ja sydämen vajaatoiminnan tautimekanismeja, uusia radiolääkeaineita ja hoitomuotoja. Avainsanat: sepelvaltimotauti, sydänlihasiskemia, sydäninfarkti, sydämen vajaatoiminta, positroniemissiotomografia (PET) 6 Table of Contents TABLE OF CONTENTS ABSTRACT.....................................................................................................................4 TIIVISTELMÄ...............................................................................................................5 ABBREVIATIONS.........................................................................................................8 LIST OF ORIGINAL PUBLICATIONS....................................................................10 1.INTRODUCTION................................................................................................... 11 2. REVIEW OF THE LITERATURE........................................................................13 2.1 Coronary artery disease......................................................................................13 2.1.1 Atherosclerotic inflammation and plaque formation................................13 2.1.2 Coronary artery stenosis and plaque rupture............................................14 2.2 Ischaemic cardiomyopathy.................................................................................14 2.2.1 Myocardial ischaemia..............................................................................14 2.2.2 Ischaemic preconditioning.......................................................................15 2.2.3 Myocardial infarction...............................................................................16 2.2.4 Cardiac remodelling.................................................................................16 2.2.5 Heart failure..............................................................................................17 2.3 Experimental models of coronary artery disease................................................18 2.3.1 Large animal models of atherosclerosis...................................................19 2.4 Experimental models of ischaemic cardiomyopathy..........................................21 2.4.1 Large animal models of chronic myocardial ischaemia...........................22 2.4.2 Large animal models of acute ischaemia-reperfusion injury and infarction.. 24 2.4.3 Large animal models of chronic myocardial infarction, remodelling and heart failure...................................................................25 2.5 Imaging of coronary artery disease and ischaemic cardiomyopathy..................28 2.5.1 Invasive coronary artery imaging.............................................................28 2.5.2 Molecular imaging of atherosclerotic inflammation................................28 2.5.3 Left ventricular function and structure.....................................................30 2.5.4 Myocardial perfusion...............................................................................30 2.5.5 Myocardial infarction and viability..........................................................31 2.5.6 PET imaging of myocardial metabolism..................................................32 2.6 Positive inotropic therapy in heart failure..........................................................32 3. AIMS OF THE STUDY...........................................................................................34 4. MATERIALS AND METHODS.............................................................................35 4.1 Coronary artery disease model (I)......................................................................35 4.1.1 Experimental protocol..............................................................................35 4.1.2 In vivo PET imaging of atherosclerotic inflammation.............................35 4.1.3 Ex vivo studies.........................................................................................35 4.2 Surgical and percutaneous myocardial infarction model (II, III).......................36 4.2.1 Experimental protocol..............................................................................36 4.2.2 PET imaging of myocardial perfusion and viability................................37 Table of Contents7 4.2.3 Left ventricular size and function (II)......................................................37 4.2.4 Myocardial oxidative metabolism and efficiency (II)..............................38 4.2.5 Tissue samples and histology...................................................................38 4.3 New myocardial perfusion tracers (IV)..............................................................38 4.3.1 Study design.............................................................................................38 4.3.2 PET imaging and kinetic modelling of [68Ga] ligands.............................39 4.3.3 Organ distribution....................................................................................39 4.3.4 In vitro binding to serum proteins............................................................39 4.4 Chronic levosimendan therapy for heart failure (V)..........................................39 4.4.1 Study design.............................................................................................39 4.4.2 Effects of chronic levosimendan therapy on MI size, LV function and remodelling........................................................................................40 4.5 Statistical analyses..............................................................................................40 5.RESULTS.................................................................................................................41 5.1 Atherosclerotic plaque inflammation (I)............................................................41 5.1.1 Ex vivo studies.........................................................................................41 5.1.2 In vivo PET imaging................................................................................41 5.2 Myocardial infarction and remodelling (II, III)..................................................42 5.2.1 Myocardial perfusion and viability..........................................................42 5.2.2 Left ventricular function (II)....................................................................43 5.2.3 Myocardial oxidative metabolism and efficiency (II)..............................43 5.2.4 Tissue samples and histology (II).............................................................43 5.3 Evaluation of new perfusion tracers (IV)...........................................................43 5.3.1 PET imaging and kinetic modelling of [68Ga] ligands.............................43 5.3.2 Organ distribution....................................................................................44 5.3.3 In vitro binding to serum proteins............................................................44 5.4 Evaluation of chronic levosimendan intervention for heart failure (V).............44 5.4.1 Effects of chronic levosimendan therapy on MI size, LV function and remodelling........................................................................................44 6.DISCUSSION...........................................................................................................46 6.1 PET imaging of early atherosclerotic lesions.....................................................46 6.2 Validation of a surgically induced model of myocardial infarction...................47 6.3 Validation of percutaneously-induced model of myocardial ischaemia and infarction.............................................................................................................48 6.4 PET imaging and kinetic modelling of [68Ga] ligands........................................48 6.5 Chronic levosimendan intervention for heart failure..........................................49 6.6 Critical evaluation of the results.........................................................................49 6.7 Future aspects.....................................................................................................50 7. SUMMARY AND CONCLUSIONS.......................................................................52 9.REFERENCES........................................................................................................55 10.ORIGINAL PUBLICATIONS................................................................................73 8 Abbreviations ABBREVIATIONS AC Adenylyl cyclase ADP Adenosine diphosphate Alpha-V beta-3 integrin αvβ3 AHA American Heart Association Akt Protein kinase B AMP Adenosine monophosphate ATP Adenosine triphosphate 18 2-[18F]-Fluoro-2-deoxy-D-glucose [ F]FDG BAPEN N,N′-bis(3-aminopropyl)ethylenediamine BAPDMEN N,N′-bis(3-aminopropyl)-dimethylenediamine CFR Coronary flow reserve COX-2 Cyclooxygenase-2 CT Computed tomography CVR Coronary vascular resistance DOTATATE 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid octreotate ECGElectrocardiography EDV End-diastolic volume EF5 2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide EF Ejection fraction ESV End-systolic volume FFA Free fatty acids FDA United States Food and Drug Administration FMISO Fluoromisonidazole FR Folate receptor Stimulatory G-protein Gs GSK-3β Glycogen synthase kinase-3β HF Heart failure HO-1 Heme oxygenase-1 IL Interleukin iNOS Inducible Nitric-Oxide Synthase IVUS Intravascular ultrasound JAK Janus kinase Adenosine triphosphate-sensitive potassium channel KATP LAD Left anterior descending coronary artery LCX Left circumflex artery LDL Low density lipoprotein LV Left ventricle MAPK MBF MCP-1 M-CSF MFR MI MMP MnSOD MPI MR MRI NF-κB NO OCT PCSK9 PET PI3K PK11195 PKA PKC PTI PTF RCA RGD ROI RPP SR SPECT STAT SUV TGF-β1 TK TLR TNFα TSPO TTC VCAM-1 VOI WMI Abbreviations9 Mitogen-activated protein kinase Myocardial blood flow Monocyte chemotactic protein 1 Macrophage-colony stimulating factor Myocardial flow reserve Myocardial infarction Matrix metalloproteinase Manganese superoxide dismutase Myocardial perfusion imaging Mannose receptor Magnetic resonance imaging Nuclear factor kappa-B Nitric oxide Optical coherence tomography Proprotein convertase subtilisin/kexin type 9 Positron emission tomography Phosphatidylinositol-3-kinase N-butan-2-yl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide Protein kinase A Protein kinase C Perfusable tissue index Perfusable tissue fraction Right coronary artery Tripeptide arginylglycylaspartic acid Region of interest Rate-pressure product Scavenger receptor Single-photon emission computed tomography Signal transducers and activators of transcription Standardised uptake value Transforming growth factor-β1 Tyrosine kinase Toll-like receptor Tumour necrosis factor alpha Translocator protein 2,3,5-triphenyl-tetrazolium chloride Vascular cell adhesion molecule 1 Volume of interest Work-metabolic index 10 List of Original Publications LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original publications I. Tarkia M, Saraste A, Stark C, Vähäsilta T, Savunen T, Strandberg M, Saunavaara V, Tolvanen T, Teuho J, Teräs M, Metsälä O, Rinne P, Heinonen I, Savisto N, Pietilä M, Saukko P, Roivainen A, Knuuti J. [18F]FDG accumulation in early coronary atherosclerotic lesions in pigs. Manuscript submitted for publication. II. Tarkia M, Stark C, Haavisto M, Kentala R, Vähäsilta T, Savunen T, Strandberg M, Hynninen V-V, Saunavaara V, Tolvanen T, Teräs M, Rokka J, Pietilä M, Saukko P, Roivainen A, Saraste A, Knuuti J. Cardiac remodeling in a new pig model of chronic heart failure: assessment of left ventricular functional, metabolic and structural changes using PET, CT and echocardiography. Journal of Nuclear Cardiology, published online 20 February 2015. III. Rissanen TT, Nurro J, Halonen PJ, Tarkia M, Saraste A, Rannankari M, Honkonen K, Pietilä M, Leppänen O, Kuivanen A, Knuuti J, Ylä-Herttuala S. The bottleneck stent model for chronic myocardial ischemia and heart failure in pigs. American Journal of Physiology - Heart and Circulatory Physiology. 2013;305(9):H1297-H1308. IV. Tarkia M, Saraste A, Saanijoki T, Oikonen V, Vähäsilta T, Strandberg M, Stark C, Tolvanen T, Teräs M, Savunen T, Green MA, Knuuti J, Roivainen A. Evaluation of 68Ga-labeled tracers for PET imaging of myocardial perfusion in pigs. Nuclear Medicine and Biology. 2012;39(5):715-723. V. Tarkia M, Stark C, Haavisto M, Kentala R, Vähäsilta T, Savunen T, Strandberg M, Saunavaara V, Tolvanen T, Teräs M, Pietilä M, Nyman L, Duvall E, Saukko P, Levijoki J, Roivainen A, Saraste A, Knuuti J. Effect of levosimendan therapy on myocardial infarct size and left ventricle function after acute coronary occlusion. A study with experimental pig model of post-infarction heart failure. Manuscript submitted for publication. The original communications have been reproduced with the permissions of the copyright holders. 1. Introduction11 INTRODUCTION Ischaemic heart disease is the leading cause of death worldwide. It is caused by coronary artery disease (CAD), which is a slowly progressing disease in which inflammatory processes together with cholesterol accumulation lead to the narrowing of the coronary artery. Narrowing of a coronary artery causes insufficient myocardial blood flow that leads to myocardial ischaemia, which is defined as reduced myocardial oxygen supply and metabolic changes due to decreased blood flow. Severely disturbed coronary blood flow or rupture of an atherosclerotic plaque resulting in intraluminal thrombosis can cause prolonged periods of ischaemia resulting in myocardial necrosis, i.e., acute myocardial infarction (MI). MI is followed by adverse cardiac remodelling including changes in size, shape, structure and physiology of the heart after myocardial injury. Chronic heart failure (HF) occurs when the heart is unable to pump enough blood to supply the blood flow needed to maintain the circulation of the body. It is an increasing health problem that is associated with high morbidity and mortality together with poor prognosis. (Finegold et al. 2013 and Sanz et al. 2013) Finding an effective animal model of atherosclerosis and myocardial ischaemia as well as MI involving reduced left ventricle (LV) function and remodelling with long survival is difficult to achieve. Several porcine models of CAD and MI exist and are used in research (Hughes et al. 2003 and Lukács et al. 2012). Still, better models for translational research are desired. Positron emission tomography (PET) imaging of myocardial perfusion has shown high performance for diagnosis of CAD (Schelbert et al. 1982 and reviewed in Danad et al. 2014). For example oxygen-15 labelled water ([15O]water) can be used in quantification of myocardial perfusion (Bergmann et al. 1984) and assessment of CAD (Reviewed in Saraste et al. 2012). Myocardial perfusion and oxidative metabolism can be measured simultaneously in a single PET study by using a carbon-11 labelled acetate ([11C] acetate) tracer. Further, calculation together with cardiac function measurements gives an estimation of cardiac efficiency (Wolpers et al. 1994 and reviewed in Knaapen et al. 2007). A fluorine-18 labelled glucose analogue 2-[18F]-Fluoro-2-deoxy-D-glucose ([18F] FDG) can be used to determine myocardial viability (Tillisch et al. 1986 and reviewed in Knuuti et al. 2002). Viable myocardium is defined as myocardium, which does not contract normally due to ischaemia, but has the potential to recover its function. New radiopharmaceuticals for cardiac PET perfusion imaging are demanded. Nitrogen-13 labelled ammonia ([13N]ammonia), rubidium-82 chloride ([82Rb]Cl) and [15O]water are the currently used PET tracers for myocardial blood flow quantification. These tracers have short physical half-lives and the production requires an expensive on-site cyclotron. A longer half-life enables centred production of the tracer and delivery for longer distances. Gallium-68 (68Ga) labelled radiopharmaceuticals have certain advantages in preclinical research including production with relatively inexpensive 12 Introduction germanium (68Ge)/68Ga generator and easy labelling of different molecules (Maecke et al. 2007). Recently, promising 68Ga-labelled tracers for myocardial perfusion imaging (MPI) have been introduced (Hsiao et al. 2009). Because of a high mortality and poor prognosis, new treatments for HF are needed. Positive inotropic therapy improves the cardiac performance including contractility leading to increased pump function. A calcium sensitiser agent levosimendan has also shown to restrict the size of MI via activation of adenosine triphosphate-sensitive potassium (KATP) channels (Kersten et al. 2000). In these studies we aimed to develop large animal models of CAD, myocardial ischaemia and MI. Multimodality imaging methods were used in validation of these experimental models together with histopathological evaluation. Also, new radiopharmaceuticals for MPI with PET were tested. Additionally, the effect of levosimendan therapy on myocardial infarct size and LV function were tested in a model of chronic HF. 2. Review of the Literature13 REVIEW OF THE LITERATURE 2.1 Coronary artery disease Atherosclerosis is a progressive inflammatory disease. Signs of atherosclerosis have been reported already in children and even in fetuses (Stary 2000 and Napoli et al. 1997). Several risk factors like hypertension, high cholesterol levels, diabetes, obesity and smoking are all related to increased risk of atherosclerosis (Dahlöf 2010). Coronary artery disease (CAD) is caused by atherosclerotic plaque formation in the coronary artery vessel wall. Formation of an atherosclerotic plaque is a complex process including inflammation, accumulation of lipids, cell death and fibrosis (Hansson et al. 2006). Narrowing of coronary arteries caused by atherosclerosis can finally lead to reduced blood flow and myocardial ischaemia. Myocardial infarction (MI) is usually caused by advanced CAD and atherosclerotic plaque rupture. Atherosclerosis usually develops slowly over years and is often diagnosed after the onset of symptoms or an acute cardiac event. Ischaemic heart disease, originating from coronary atherosclerosis, is the leading cause of death worldwide (Finegold et al. 2013). 2.1.1 Atherosclerotic inflammation and plaque formation Atherosclerotic plaques develop in the arterial wall in response to local endothelial cell dysfunction and local inflammation (Libby 2012). Cardiovascular risk factors like hypertension, hypercholesterolemia, diabetes and smoking can cause endothelial cell dysfunction, which leads to the upregulation of adhesion molecules and inflammatory cell recruitment (Libby et al. 2005). Atherosclerosis is initiated by the accumulation of lowdensity lipoprotein (LDL) particles into the vessel wall (Skålén et al. 2002). Oxidation of LDL leads to the expression of leukocyte adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1), which lead to the infiltration of monocytes and T lymphocytes and, in combination with leukocytes, causes the secretion of chemokines such as tumour-necrosis factor alpha (TNFα), interleukins (ILs), monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinases (MMPs) (Libby 2002). Monocyte differentiation into macrophages is induced by macrophage-colony stimulating factor (M-CSF). Subsequently, scavenger receptors (SRs) and Toll-like receptors (TLRs) are upregulated. Formation of foam cells is mediated by SR, whereas TLR initiated signal cascades lead to the activation of inflammation (Figure 1) (Yan et al. 2007). Inflammatory processes and accumulation of lipid droplets lead to initial atherosclerotic changes called fatty streaks and later formation of advanced plaques and atheromas (Hansson et al. 2006). 14 Review of the Literature Blood monocyte Adhesion molecule VCAM‐1 Arterial lumen Arterial intima SR T lymphocyte TLR Oxidised LDL Monocyte becoming intimal macrophage Macrophage Foam cell Chemokines (e.g., TNFα, ILs, MCP‐1, MMPs) Figure 1. Inflammatory processes related to atherosclerosis. 2.1.2 Coronary artery stenosis and plaque rupture The earliest stage of progressive atherosclerosis is intimal thickening, which is defined as an increased number of intimal smooth muscle cells. This continuous inflammatory process leads to the formation of a fibroatheroma, a lesion with a necrotic core and an overlying fibrous cap. Progressive atherosclerosis leads to more advanced plaques and narrowing of vessel lumen. Over time, atherosclerosis can cause myocardial ischaemia by the narrowing of the vessel lumen or plaque rupture. Rupture often develops a luminal thrombus, which is a common cause of acute MI and sudden death (Virmani et al. 2000). 2.2 Ischaemic cardiomyopathy 2.2.1 Myocardial ischaemia Myocardial ischaemia occurs when the supply of oxygen no longer meets the requirement of myocardial demand. Myocardial ischaemia is caused by the limited amount of blood supply into myocardial tissue. The blood supply of heart is organised through coronary circulation and CAD is the most common cause of chronic myocardial ischaemia or infarction (Sanz et al. 2013). Insufficient blood flow can lead to regional myocardial dysfunction and later on to cardiac remodelling and HF. Under normal aerobic conditions, fatty acids supply 60% to 90% of the energy for adenosine triphosphate (ATP) synthesis. Approximately 10% to 40% of the energy comes from oxidation of pyruvate and only small amount (3%) is derived by glycolysis (Stanley 2004). Partial reduction in coronary blood flow shifts cardiomyocytes to utilise Review of the Literature15 more glucose. Glucose metabolism needs 10% less oxygen than the oxidation of fatty acids. Still, the most of the energy (50-70%) is derived from the oxidation of fatty acids (Figure 2) (Stanley 2001). Critical myocardial ischaemia results in increase of anaerobic glycolysis leading to production of lactate. The myocyte swells due to imbalance of intracellular homeostasis, which can lead to ischaemic cell death and a decrease in contractile work. Aerobic conditions Ischaemic conditions Glycolysis Fatty acids 3% 60-90% Lactate Pyruvate Glycolysis ATP ADP Pyruvate Lactate 10-40% Contraction ATP Fatty acids 50-70% 30-40% O2 Contraction ATP Mitochondrion ADP Mitochondrion ADP Figure 2. Myocyte energy metabolism under normal aerobic and ischaemic conditions. 2.2.2 Ischaemic preconditioning Ischaemic preconditioning refers to the myocardial protection occurring after brief periods of sublethal ischaemia. It has been linked to reduced MI size and increased survival. Protective effects start immediately after coronary occlusion following reperfusion (first window) (Murry et al. 1986). The first window of protection is activated through opioid, adenosine, bradykinin or alpha1-adrenergic receptors lasting 4 to 6 hours (Figure 3) (Rodrigo et al. 2008). Activation of protein kinase C (PKC) leads to activation of mitochondrial KATP channel. The second window of protection has been demonstrated 24 hours after the preconditioning lasting up to 72 hours (Buja 2005 and Das et al. 2008). The second window is activated through nuclear factor kappa-B (NF-κB) (Figure 3). Preconditioning reduces the energy demand of the myocardium leading to preserved myocardial function and reduction of arrhythmias (Hagar et al. 1991). The exact mechanisms of this preconditioning effect remain unclear. 16 Review of the Literature Ischaemia/reperfusion Receptor activation (opioid, adenosine, bradykinin) PI3K Cardiac nerves (α1‐adrenergic receptors) 2+ PKC Endotoxins PKC, MAPK TK, JAK/STAT Ca Akt Exercise NO NF‐κB GSK‐3β Mitochondrial KATP channel Transition pore Oxygen radicals First window iNOS, COX‐2, MnSOD HO‐1, aldose reductase Second window Figure 3. Mechanisms of the first and second window of ischaemic preconditioning. 2.2.3 Myocardial infarction Myocardial infarction (MI) is caused by severe and prolonged ischaemia. The existence of coronary collateral flow is one determinant of infarct size. Necrosis occurs first in the subendocardial region. The necrotic area expands progressively transmurally, i.e., affecting the entire thickness of the wall within time (Reimer et al. 1979). The major cause of acute MI is coronary atherosclerosis with luminal thrombus. MI without atherosclerosis is rare. A prolonged period of insufficient blood flow leads to MI. Wound-healing involves fibrosis formation and MI scarring. Inflammatory cells, mainly macrophages, initially infiltrate into the necrotic myocardium. Interstitial fibroblasts are transformed into myofibroblasts by transforming growth factor-β1 (TGF-β1). Myofibroblasts express alpha-type smooth muscle actin and contain angiotensin-converting enzyme and matrix metalloproteinases. TGF-β1 and angiotensin II are responsible for accumulation of collagen. Changes in the myocardial extracellular matrix are very rapid and formation of collagen in the infarcted area is visible two to three days after MI. Infarcted myocardium thins and is replaced with non-functioning collagen-rich scar tissue (Swynghedauw 1999 and Bujak et al. 2007). 2.2.4 Cardiac remodelling Myocardial remodelling includes all those changes that occur after MI to compensate for the loss of a functional myocardium. Remodelling is initiated by increased mechanical Review of the Literature17 stretch due to MI scarring. A major determinant for infarct expansion and remodelling is the transmural extent of necrosis. Complex neurohumoral remodelling processes usually lead to viable cardiac tissue hypertrophy, fibrosis and cell death. After MI, systolic function will be impaired due to the loss of contractile myocardium. This leads to increased end-systolic volume, increased heart size and increased diastolic filling pressure (Swynghedauw 1999). Elevated myocardial wall stress will be normalised by hypertrophy according to Laplace’s law. The wound healing and remodelling processes after MI involves both the infarcted and non-infarcted myocardium. Fibrosis is more related to scar formation when it is replacing necrotic tissue but also to remodelling occurring interstitially in non-infarcted remote tissue. Fibrosis in the remote myocardium can be induced by vasoactive peptides and hormones like angiotensin II and by mechanical pressure overload (Heusch et al. 2014). 2.2.5 Heart failure HF occurs when the heart is unable to pump enough blood to maintain normal circulation. It is a consequence of an abnormality in cardiac structure, function, rhythm or conduction. The most common causes of heart failure are myocardial infarction and hypertension (McMurray et al. 2005). HF can appear acutely especially in the case of MI. Most clinical signs are non-specific. Electrocardiography (ECG) can be used to measure pathological changes in the electrical conduction system of the heart. Echocardiography is widely used to demonstrate cardiac dysfunction. Other imaging modalities, such as computed tomography (CT), PET and magnetic resonance imaging (MRI) are used increasingly in the evaluation of HF. Despite recent advances in pharmacological and device therapies, prognosis of HF is often poor: 30 to 40 percent of patients die within a year and 60 to 70% die within 5 years of diagnosis (McMurray et al. 2005). Energy metabolism is disturbed in HF. Contractile performance is decreased together with reduction of ATP, phosphocreatine and creatine kinase concentration (Liao et al. 1996). Oxygen deprivation results in impaired relaxation and contraction. Stunning can be present after prolonged ischaemia as decreased contraction persisting for hours after the return of blood flow. During hibernation, blood flow is chronically limited and the myocardium contraction fails. Inadequate blood flow leads to MI, LV remodelling and further HF (Vanoverschelde et al. 1993). The oxygen requirement of the heart can be increased in HF due to increased cardiac work, myocardial mass, increased myocardial wall stress and wasted contractile energy. After MI, neurohumoral activation and myocardial load and stretch are increased. It leads to myocardial growth response inducing myocardial hypertrophy and myocardial remodelling. The final outcome is an increased cardiac energy expenditure and myocardial cell death through apoptosis and necrosis leading to HF (Figure 4) (Heusch et al. 2014). 18 Review of the Literature Myocardial infarction Load, stretch Cardiac output Load, stretch Neurohumoral activation Myocardial growth response Cardiac energy expenditure Myocardial hypertrophy Cell elongation Remodelling Apoptosis Myocyte necrosis Myocardial cell death Heart failure Figure 4. Mechanisms leading to myocardial remodelling and HF. 2.3 Experimental models of coronary artery disease Atherosclerosis research using human samples is difficult because of the slow and unnoticeable development of the disease. Tissue samples are also difficult or impossible to obtain from humans. Therefore, animal models are needed. Several small animal atherosclerosis models have been developed by genetic engineering, e.g., modifying genes coding the LDL receptor or apolipoproteins (Heinonen et al. 2007). Despite several benefits, small animals have some restrictions like small size and differences in anatomy and physiology compared to humans. Large animal models are better suitable for studying coronary arteries, which are difficult or even impossible to study by using small animals. Also, achieving a more human-like size, anatomy and physiology is critical in translational research where basic research advances can be easily transferred onto a clinical stage. Rabbit models have been widely used in cardiovascular research. Watanabe rabbits have a high serum level of LDL-cholesterol and are prone to produce atherosclerosis within a reasonable time when fed with an atherogenic diet (Aliev et al. 1998). A pig model Review of the Literature19 is considered very appropriate because the anatomy and physiology of the heart and coronary vasculature are very similar to that of humans (Bertho et al. 1964, Kamimura et al. 1996, Kassab et al. 1997 and Swindle et al. 2012). The anatomy of the pig heart is nearly similar than that of the human heart. The LV wall is thicker than in human heart. Only two pulmonary veins drain into the left atrium. The left azygous vein drains into the coronary sinus, but otherwise the coronary circulation is identical with humans (Weaver et al. 1986, Crick et al. 1998 and Swindle et al. 2012). Pig models are used for characterising the role of monocytes during early atherosclerosis as well as smooth muscle and endothelial cell proliferation in plaque development (Gerrity 1981a, Gerrity 1981b and Gerrity et al. 1985). Atherosclerotic lesions are mainly located similarly as in humans in the proximal part of coronary arteries (Gerrity et al. 2001 and Thim et al. 2010). The morphology and progression of plaques in pig are similar to that seen in humans. Also, blood glucose, cholesterol and lipoprotein levels and metabolism are similar to those of humans. 2.3.1 Large animal models of atherosclerosis A large animal model mimicking human-like coronary atherosclerosis is lacking, but highly needed for translational research approaches. Farm pigs with familial hypercholesterolemia develop atherosclerosis in two to three years (Thim et al. 2010). In that case, animals are usually weighing more than 200 kg and the extensive housing costs of pigs are expensive. Atherosclerosis can be induced by different techniques. The most relevant techniques with characteristics are presented in Table 1. Normally farm pigs are naturally very resistant to develop inherent atherosclerosis. Early atherosclerotic lesions, mainly fatty streaks, are possible to achieve by hypercholesterolemia that is induced by an atherogenic diet containing high amounts of saturated fatty acids and cholesterol. Blood total cholesterol level increases from a normal level of 2 mmol/L to 5 mmol/L with a diet containing 2 percent cholesterol and 17 percent to 20 percent fat (Artinger et al. 2009 and Pueyo Palazón et al. 1998). With additional sodium cholate in the diet (1.5%), it is possible to achieve more advanced atherosclerotic plaques including fibroatheroma and cholesterol crystals with 24 to 41 percent coronary artery stenosis (Thorpe et al. 1996). The formation of atherosclerosis can be accelerated by inducing diabetes by using streptozotocin to destroy insulin-secreting beta cells in pancreatic tissue and feeding pigs an atherogenic diet containing a high amount of cholesterol (1.5%) and lard (15%) (Gerrity et al. 2001). In that model both plasma glucose and cholesterol levels are possible to maintain near 20 mmol/L leading to more advanced atherosclerotic plaque formation including a well-developed fibrous cap overlying necrotic lipid core and coronary artery stenosis as high as 98 percent (Gerrity et al. 2001). Plaque development can also be controlled in these models using statin or angiotensin converting enzyme inhibitor therapy (Chatzizisis et al. 2009). 20 Review of the Literature Genetic models have also been demonstrated. In a minipig model that has a LDL receptor mutation, atherosclerosis was successfully induced by an atherogenic diet and mechanical injury of vessel wall (Thim et al. 2010). Pigs having mutant alleles for apolipoprotein B develop spontaneous hypercholesterolemia. Early atherosclerotic changes are visible during the first year. At two years of age, advanced plaques with a necrotic core with fibrous cap and stenotic lesions exist. By three years of age, complicated lesions and rupture are common. (Prescott et al. 1991) Rapacz familiar hypercholesterolemia pigs develop atherosclerotic lesions mainly in peripheral arteries and are thus more relevant in studies related to peripheral arterial disease (Bahls et al. 2011). D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene lead to autosomal dominant hypercholesterolemia and atherosclerosis in humans. Pigs with DNA transposition of a human PCSK9 gain-of-function mutant represent an atherosclerosis model of familial hypercholesterolemia. (Al-Mashhadi et al. 2013) Ossabaw swine represents a metabolic disease model representing increased blood glucose and impaired insulin sensitivity. Early atherosclerotic changes develop over time. (Dyson et al. 2006) To accelerate CAD development, an oversized angioplasty balloon can be used to induce mechanical vessel wall injury. Histological changes are usually occur at the very early stage, but also advanced atherosclerotic lesions including neointima expansive remodelling and fibroatheroma have been observed in these models (Thim et al. 2010). Coronary occlusion can be induced with a mechanical occluder to simulate plaque rupture, a complication of atherosclerosis. An ameroid constrictor has been used in atherogenic diet fed pigs in studies related to mechanisms after the rupture, e.g., collateral formation (Matyal et al. 2012). The limitations of pig models of CAD include the lack of spontaneous development of atherosclerosis and that formed plaques remain at the initiation state without stenosis and rupture leading to MI. Also, atherosclerosis can only be induced over prolonged periods of time, which leads to increased costs and difficulties when handling heavy animals. Review of the Literature21 Table 1. Characteristics of pig models of atherosclerosis. Technique Characteristics Hypercholesterolemia Early atherosclerotic lesions, fatty streaks, intimal thickening, low-grade stenosis Hypercholesterolemia + hyperglycemia Genetic modifications Metabolic disease Vessel wall injury Reference Artinger et al. 2009, Turk et al. 2005, Pueyo Palazón et al. 1998, Thorpe et al. 1996, Kim et al. 1993 and Shimokawa et al. 1988 Gerrity et al. 2001, Wilensky et al. 2008, LiFeng Zhang et al. 2003, Advanced atherosclerotic lesions, necrotic Hamamdzic et al. 2010, Mohler et al. lipid core, well-developed fibrous cap, high2008, Artinger et al. 2009, McDonald grade stenosis et al. 2007, Dixon et al. 1999 and Chatzizisis et al. 2009 Prescott et al. 1991, Hasler-Rapacz et al. 1998, Hasler-Rapacz et al. 1995, Spontaneous hypercholesterolemia, fatty Prescott et al. 1995, Tellez et al. 2010, streaks, advanced plaques containing necrotic Bahls et al. 2011, Granada et al. 2011, core, calcification, neovascularization, Thim et al. 2010, Al-Mashhadi et al. hemorrhage and rupture. 2013 and Thim, Hagensen, WallaceBradley, et al. 2010 Increased blood glucose, insulin resistance, Dyson et al. 2006, Neeb et al. 2010 early atherosclerotic changes and Kreutz et al. 2011 Tellez et al. 2011, Worthley et al. 2000, Early atherosclerotic-like lesions, eccentric Worthley et al. 2000, Mihaylov et al. fibrocellular plaques, increased intima-media 2000, Carter et al. 1996, Shimokawa et ratio al. 1985 and Thim et al. 2010 Matyal et al. 2012, Matyal et al. Regional ischaemia/infarction 2013, Lassaletta et al. 2012 and Robich et al. 2011 Hypercholesterolemia + mechanical coronary occlusion Intramural injection of cholesteryl Lipid-containing inflammatory lesions linoleate Granada et al. 2005 2.4 Experimental models of ischaemic cardiomyopathy The most used experimental models for ischaemic cardiomyopathy are mouse and rat. Rodents have many advantages like the availability, cost-effectiveness, easy handling and transgenic strains. However, there are significant differences in the anatomy and physiology of the heart and circulatory system between rodents and humans. Dog models of myocardial ischaemia have been successfully used to study mechanisms behind MI and later myocardial remodelling and HF (Reimer et al. 1977, Reimer et al. 1979, Przyklenk et al. 1986 and Jugdutt et al. 1979). The pre-existing collateral circulation makes it difficult to induce large myocardial ischaemia or infarction in dogs (Jugdutt et al. 1979). The absence of an existing collateral network enables studies related to myocardial adaptation to ischaemia in pigs. The pig heart and circulatory system is very close to that of humans and is therefore suitable as a humanoid model of heart diseases (Swindle et al. 2012). Several pig models having different cardiomyopathies have been introduced and can be used in studies of mechanisms of heart failure and cardiac remodelling. Pressure 22 Review of the Literature overload induced by aortic stenosis can be used to cause LV hypertrophy through adaptive mechanisms that normalise to increased LV wall stress (Gelsomino et al. 2013). This situation contributes to the preservation of normal LV function and excessive collagen accumulation (Dixon et al. 2009). LV hypertrophy is a significant contributor of HF. Reduced LV relaxation and filling together with increased LV stiffness creates pressure overload models well-suited for the investigation of HF. Volume overload models can be used to mimic the clinical situation of mitral valve regurgitation. A disease model can be induced by chordal rupture of the mitral valve. Volume overload produces cellular level abnormalities causing decreased myocyte contractile function finally leading to HF. (Dixon et al. 2009) Doxorubicin-induced HF is another model to study global HF. Intravenous injection of cardiotoxic doxorubicin rapidly progresses to systolic and diastolic dysfunction and ECG abnormalities (Torrado et al. 2011). Dilated cardiomyopathy is defined as LV dilatation resulting in increased LV wall stress. Pacemaker-induced chronic tachycardia has been used to induce dilated cardiomyopathy and HF since 1962 (Dixon et al. 2009). Nowadays modern telemetry systems can be used in these models for chronic monitoring of changes in several hemodynamic parameters simultaneously (Choy et al. 2014). 2.4.1 Large animal models of chronic myocardial ischaemia Myocardial ischaemia is possible to induce in several ways by causing limited blood flow in coronary artery with stenosis. In surgical open-chest models, coronary artery stenosis is induced by an occluder placed around the coronary artery. An ameroid constrictor placed around the coronary artery induces gradually an increasing reduction in blood flow leading finally to total occlusion of coronary artery. An adjustable occluder or shunt applications can be used to obtain different levels of stenosis. Mechanically working hydraulic, screw type or inflatable occluders can be used to achieve adjusted blood flow. For studies concerning myocardial angiogenesis, energy metabolism and other cellular mechanisms under chronic myocardial ischaemia, a constantly limited blood flow is desired. Fixed-diameter occluders produce constant stenosis and permanently limited blood flow. Delran stenosis, C-shape occluder or ligations are mostly used to produce fixed stenosis. Myocardial ischaemia is possible to induce percutaneously by using invasive angiographic methods. In closed-chest models, ischaemia can be induced with an angioplasty balloon, intracoronary flow-reducer, stent or by vessel wall injury. An angioplasty balloon can be used to create acute myocardial ischaemia whereas chronic ischaemia is possible to induce by stents or vessel wall injury. Chronic myocardial ischaemia leads to deteriorated LV function due to decreased contractility. Regional ischaemia is often seen as decreased wall motion, perfusion and Review of the Literature23 oxygen consumption. Apoptosis, glucose utilisation and lactate accumulation are usually increased. Different techniques with characteristics and selected references are listed in Table 2. Changes in vessel geometry and intracoronary artificial occluders predispose to thrombosis leading to acute MI. Anticoagulant therapy has been successfully used to prevent premature coronary obstruction. Table 2. Characteristics of pig models of chronic myocardial ischaemia. Technique Ameroid constrictor Carotid coronary shunt Characteristics Reference Reduced left ventricular function, reduced Caillaud et al. 2010 and Giordano et blood flow al. 2013 Increased K+ concentration, decreased pH, ST Watanabe et al. 1987 changes Fallavollita et al. 1999, Fallavollita et al. 1997, Fallavollita, Lim, et al. 2001, Fallavollita, Logue, et al. 2001, Regional LV motion abnormality, reduced Fallavollita 2000, Fallavollita 2002, Fixed-diameter resting perfusion, hibernating myocardium, Fallavollita et al. 2002, Fallavollita et occluder reduced ejection fraction (EF), increased al. 2005, Lim et al. 1999, Hardt et al. apoptosis, reduced flow reserve 2001, McFalls et al. 2006, McFalls et al. 1997, Mills et al. 1994, Bloor et al. 1984, Ishikawa et al. 2011 and Heilmann et al. 2006 Regional LV motion abnormality, decreased Thomas et al. 1999, Kim et al. 2001, wall-thickening, reduced perfusion, impaired St. Louis et al. 2000, Hughes et al. Ca2+ handling, depletion of contractile materi- 2001, Chen et al. 1996, Chen et al. Adjustable al, glycogen accumulation, increased amount 1997, Lai et al. 2000, Liedtke et al. occluder of mitochondria, decreased oxygen consump- 1995, Chen et al. 1997, Liedtke et al. tion, increased glucose consumption, lactate 1994, Bolukoglu et al. 1992, Cason et production, reduced left ventricular EF, inal. 1991, Galiuto et al. 2002 and Sascreased apoptosis, decreased flow reserve sen et al. 1988 Angioplasty Bamberg et al. 2012 and Mahnken et Regional perfusion defect balloon al. 2010 Gewirtz et al. 1981, de Groot et al. 2011, von Degenfeld et al. 2003, Intracoronary flow- Regional perfusion defect, impaired myocar- Johnson et al. 1998, Gewirtz, Brautireducer dial function, increased glucose utilisation gan, et al. 1983, Gewirtz, Williams, et al. 1983, Kraitchman et al. 2000 and Kraitchman et al. 2002 Wu et al. 2011, Wu et al. 2010, Bito et Decreased ejection fraction, hibernation, deal. 2004, Szilárd et al. 2000 and HorCopper stent creased wall-thickening stick et al. 2009 In-stent neointimal Hemetsberger et al. 2008 and Johnson Over 40% stenosis after 1-month follow-up hyperplasia et al. 2000 Grinstead et al. 1994, Schwartz et al. Coronary artery Neointimal thickening, over 50% stenosis 1990, Schwartz et al. 1992 and Anderinjury after 1-month follow-up sen et al. 1996 24 Review of the Literature 2.4.2 Large animal models of acute ischaemia-reperfusion injury and infarction Myocardial injury leading to impaired LV function and HF can be induced by prolonged episodes of myocardial ischaemia followed by reperfusion. Temporal occlusion by angioplasty balloon is the most commonly used method to occlude coronary artery completely but only for a short period of time. Regional ischaemia occurs immediately after inflating the balloon. Deflating the balloon leads to reperfusion. Severity of ischaemia-reperfusion injury depends on the duration of ischaemia following reperfusion. Different techniques and characteristics are presented in Table 3. A cardiopulmonary bypass model can be used to mimic the situation in open heart surgery or transplantation. An animal is first connected to a heart-lung machine. The heart is arrested with cardioplegia and the remaining body is perfused by using cardiopulmonary bypass. Global myocardial ischaemia-reperfusion injury can be studied after weaning from a heart-lung machine. Despite that the heart is wellprotected with cardioplegia, damage of muscular fibres, mitochondrial swelling and intracellular oedema can be observed with transmission electron microscopy (Hong et al. 2013). Hemodilution, volume loading as well as cytokine and catecholamine surge are often induced after cardiopulmonary bypass and reperfusion (Olson et al. 2012). The clinical situation with coronary artery occlusion following the resolution of clots and reperfusion can be modelled by regional ischaemia-reperfusion injury induced with temporal occlusion of the coronary artery surgically with clamping and ligation or percutaneously with angioplasty balloon. An ischaemic period of 30 to 120 minutes seems to be well-tolerated. Ventricular extrasystoles, non-sustained tachycardia and ventricular fibrillation can occur during ischaemia. Myocardial stunning is related to inadequate contractility of cardiomyocytes after an ischaemic condition following reperfusion and is possible to study with these models. MI is usually observed in these models without protective cardiac arrest. Cardiac arrest has been caused also by using alternative methods like asphyxia induced by endotracheal tube clamping, electric fibrillation or placement of intracoronary ball. Review of the Literature25 Table 3. Characteristics of pig models of acute ischaemia-reperfusion injury and infarction. Technique Characteristics Cardiopulmonary bypass Decreased LV contractility, function and blood pressure, increased myocyte apoptosis, coronary blood flow and troponin levels, myofibril damage and neutrophil infiltration Coronary clamping Stunning, decreased LV function and blood pressure, increased heart rate, MI Coronary ligation Decreased LV function, increased apoptosis and oxidative stress, MI Balloon occlusion Decreased LV contractility, function, increased apoptosis, MI Asphyxia Decreased LV contractility and function Electric fibrillation Elevated cardiac enzymes Intracoronary ball Elevated cardiac enzymes, MI Reference Hong et al. 2013, Olson et al. 2012, Shinohara et al. 2011, Salminen et al. 2011, Abdel-Rahman et al. 2009, Banz et al. 2008, Jormalainen et al. 2007, Malmberg et al. 2006, Vähäsilta et al. 2005 and Lim et al. 2005 Díez et al. 2013, Aarsæther et al. 2012 and Sala-Mercado et al. 2010 Xiang-dong Li et al. 2013, Doganci et al. 2012, Meyer et al. 2013, Skyschally et al. 2013, Chinda et al. 2013, Kanlop et al. 2011, Arslan et al. 2012, Gelsomino et al. 2012, Gelsomino et al. 2011, Oyamada et al. 2010, Sodha et al. 2009, Osipov et al. 2009, Metzsch et al. 2006 and Garcia-Dorado et al. 1987 Hashizume et al. 2013, Duran et al. 2012, Ogura et al. 2012, Wheeler et al. 2012, Barallobre-Barreiro et al. 2012, Lu et al. 2013, Wojakowski et al. 2012, Chatziathanasiou et al. 2012, Bhindi et al. 2012, Poulsen et al. 2011, Dash et al. 2011, van der Pals et al. 2010, Wiggers et al. 1997, Silva et al. 2009, Boekstegers et al. 2002 and Hinkel et al. 2013 Lin et al. 2013 Bertsch et al. 2001 and Bertsch et al. 2000 Näslund, Häggmark, Johansson, Marklund, et al. 1992 and Näslund, Häggmark, Johansson, Pennert, et al. 1992 2.4.3 Large animal models of chronic myocardial infarction, remodelling and heart failure A large animal model of chronic MI and HF is crucial in translational research to study complex mechanisms underlying ischaemic heart diseases. Based on literature searches, the most common way to induce MI is to use an angioplasty balloon to occlude coronary artery totally for a temporal period of time following reperfusion. Coronary artery ligation, ameroid constrictor and embolisation are also widely used methods. Different techniques and characteristics are presented in Table 4. MI can be induced percutaneuosly by angioplasty balloon. The coronary artery is catheterised under X-ray angiography. An angioplasty balloon is placed into the coronary artery and inflated to occlude the vessel totally. After the desired occlusion time, the balloon is deflated and removed. The severity of MI depends on the occlusion 26 Review of the Literature time and the balloon location. A 60-minute occlusion time produces a large transmural MI. Occlusion of 30 minutes does not produce myocardial necrosis and a 45-minute occlusion produces only a small necrotic area (Garcia-Dorado et al. 1987). Ischaemic preconditioning can be induced with repetitive balloon occlusions (Yang et al. 2011). Coronary artery ligation and ameroid constrictor placement requires surgery. Usually thoracotomy is enough for accessing coronary arteries. A direct view of the left anterior descending coronary artery (LAD) is possible to achieve by dissecting skin and muscle between the third and fourth ribs. A spreader needs to be used to separate the ribs. After dissecting pericardium, LAD is visible. Permanent ligation of the coronary artery leads to acute MI following LV remodelling and worsening of LV function (Huang et al. 2010 and J. Zhang et al. 1996). Infarction can also be induced by temporal coronary artery ligation following reperfusion (So et al. 2012). An ameroid constrictor placed around a coronary artery induces gradual occlusion leading usually to MI. Complete coronary occlusion is necessary in research related to MI and for example, collateral growth. Induction of MI by inducing embolisation with thrombogenic material like embolisation coils, intracoronary ethanol or a gelatine sponge leads to very similar MI and later changes than coronary ligation (Gálvez-Montón et al. 2014). The size of MI should be large enough to induce remodelling processes. Distal and midpoint ligation of LAD leads to a MI size of 10 percent and 14.9 percent, respectively (Munz et al. 2011). MI sizes of 12.8 percent and 23.8 percent are reported to be achieved by ligating one-third of the LAD from the apex and below the second diagonal branch, respectively (Huang et al. 2010). Over 25 percent MI of LV is possible to achieve with proximal occlusion of LAD whereas proximal left circumflex artery (LCX) occlusion induces MI covering 20 percent of LV (Teramoto et al. 2011, Munz et al. 2011 and Roth et al. 1987). High mortality rates have been reported if the MI size exceeds 25% of the LV (Kamimura et al. 1996). Sudden cardiac deaths are reported to be in relation to fatal arrhythmias due to intolerance of ischaemia (Fallavollita et al. 2005). The ligation of a distal part of coronary artery demonstrates reasonable survival rates but only small MI. Gradual coronary artery occlusion induced using an ameroid constrictor has interestingly led to very small MI and an aggressive collateral development may be the cause (Roth et al. 1987). Combining the distal coronary ligation and proximal ameroid constrictor demonstrates large MI (>25% of LV) with clear signs of remodelling. End-diastolic and end-systolic volumes were markedly larger and EF lower in MI group when compared to controls. Hypertrophy and fibrotic changes can visualised by histology in MI group. Also, a survival rate at the 4-month time point was reported to be as high as 75 percent (Teramoto et al. 2011). A study configuration with only a proximal ameroid constrictor produced a survival rate of 30 percent. The pig heart is very sensitive to acute ischaemia Review of the Literature27 and sudden cardiac deaths can occur (Fallavollita et al. 2005). The mechanism of inhibition of sudden cardiac death with distal coronary ligation is not well-known. One hypothesis is that distal ligation provides a preconditioning effect through ischaemia and small MI as demonstrated by several studies (Murry et al. 1986, Kuzuya et al. 1993 and Hagar et al. 1991). Also, gradual occlusion may adapt the myocardium to tolerate ischaemia. A chronic HF model with impaired LV systolic function including LV dilatation, reduced EF and cellular evidence of LV remodelling offers new possibilities in translational research. Table 4. Characteristics of pig models of chronic MI, remodelling and HF Technique Characteristics Coronary ligation Transmural MI, impaired LV function, LV dilatation, increased apoptosis Ameroid constrictor Transmural MI, impaired LV function, LV dilatation, impaired LV wall motion and increased myocyte hypertrophy and interstitial fibrosis in the non-infarcted remote tissue, rapid collateral development Angioplasty balloon Transmural MI, impaired LV function, LV dilatation Embolisation Transmural MI, impairer LV function and wall motion, LV dilatation Reference Garcia-Dorado et al. 1987, Jiang et al. 2014, Zhu et al. 2013, Prescimone et al. 2013, Munz et al. 2011, Kuster et al. 2011, So et al. 2012, Qu et al. 2012, Sahul et al. 2011, Weiss et al. 2010, Huang et al. 2010, Cho et al. 2008 and Shuros et al. 2007 Giordano et al. 2013, Kawamura et al. 2012, Shudo et al. 2011, Teramoto et al. 2011, Barandon et al. 2010, Schneider et al. 2010, Tuzun et al. 2010, Pätilä et al. 2009, Christian et al. 2008, Ikonen et al. 2007 and Roth et al. 1987 Xiaorong Li et al. 2014, Tanaka et al. 2014, Sheriff et al. 2014, Vilahur et al. 2014, Varga-Szemes et al. 2014, Pavo et al. 2014, van Hout et al. 2013, Koudstaal et al. 2013, Yan Chen et al. 2013, Duran et al. 2012, Yang et al. 2011, Pleger et al. 2011, Angeli, Amabile, Shapiro, et al. 2010, Angeli, Amabile, Burjonroppa, et al. 2010, Lautamäki et al. 2009, Pérez de Prado et al. 2009, Holz et al. 2009, Brødløs et al. 2009, Krombach et al. 2005, Garcia-Dorado et al. 1987 and Abegunewardene et al. 2009 Gálvez-Montón et al. 2014, GálvezMontón et al. 2013, Biondi-Zoccai et al. 2013, Fish et al. 2013, Saeed et al. 2013, Song-Yan Liao et al. 2010, Peukert et al. 2009, Dib et al. 2006, Cui et al. 2005, Dogné et al. 2005, Waksman et al. 2004, Reffelmann et al. 2004, Sakaguchi et al. 2003, Crisóstomo et al. 2013, Weon Kim et al. 2011, Joudinaud et al. 2005 and Ohtsuka et al. 2003 28 Review of the Literature 2.5 Imaging of coronary artery disease and ischaemic cardiomyopathy 2.5.1 Invasive coronary artery imaging Coronary artery stenosis, plaques and their progression can be evaluated with invasive angiography combined with intravascular ultrasound (IVUS) and optical coherence tomography (OCT). Calcified plaques, shear stress as well as atheroma volume and fibrous cap thickness can be assessed with OCT (Sanz et al. 2013). 2.5.2 Molecular imaging of atherosclerotic inflammation Several pathological processes related to atherosclerosis can be assessed with PET imaging. Plaque formation is a response to the inflammatory process. Macrophages are a good imaging target (Rudd et al. 2002). Also, neoangiogenesis, hypoxia and microcalcification are associated with advanced plaque formation and are good targets for imaging purposes (Tarkin et al. 2014). [18F]FDG is a radiolabelled glucose analogue, which is widely used in PET imaging of metabolic activity. [18F]FDG is taken up by the cells using glucose transporters. Phosphorylated [18F]FDG is trapped inside the cell. Clearance from blood circulation is rapid and trapped tracer accumulation can be visualised even with a low background if PET imaging is performed for some time after the tracer injection. Because of high glucose metabolism of myocardium, the analysis of the [18F]FDG uptake in coronary arteries can be difficult due to high background activity. A low-carbohydrate, high-fat meal prior to scanning has been introduced to suppress myocardial uptake (Wykrzykowska et al. 2009). Increased vascular [18F]FDG accumulation indicates increased macrophage activity in atherosclerotic inflammation (Figure 5) (Tarkin et al. 2014). Mannose receptors (MRs) are expressed by macrophages in high-risk plaques and are suggested to serve as more specific targets of imaging tracers (Figure 5). 18F-labelled mannose has successfully been used in the visualisation of atherosclerotic lesions (Tahara et al. 2014). Overexpression of somatostatin receptors in activated macrophages can be visualised with the [68Ga]DOTATATE (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid octreotate) tracer (Figure 5) (Rominger et al. 2010). The translocator protein (TSPO) ligand [11C]PK11195 (N-butan-2-yl-1-(2-chlorophenyl)N-methylisoquinoline-3-carboxamide) accumulates during vascular inflammation through increased TSPO expression in activated macrophages (Figure 5) (Laitinen et al. 2009). Folate receptors (FRs) are expressed by activated macrophages (Figure 5). Increased uptake of folate receptor targeting tracers in atherosclerotic plaques has been shown in recent studies (Ayala-López et al. 2010 and Jager et al. 2014). Scavenger receptors (SRs) are related to the macrophage differentiation into foam cells. Radiotracers targeting scavenger receptor may help to visualise foam cells in active atherosclerotic lesions (Bigalke et al. 2014). Review of the Literature29 Increased expression of different adhesion molecules are related to atherosclerotic inflammation and, for example, P-selectin and VCAM-1 are possible targets for imaging tracers (Figure 5) (Nakamura et al. 2013 and Nahrendorf et al. 2009). Choline takes part in cell membrane formation. Choline is believed to be accumulated in activated macrophages and incorporated into cell membranes after phosphorylation by choline kinase (Figure 5). Increased uptake of [11C]choline and [18F]choline in atherosclerotic plaques has been demonstrated (Laitinen et al. 2010 and Matter et al. 2006). Hypoxia plays an important role in atherosclerotic plaque formation (Figure 5). [18F] fluoromisonidazole ([18F]FMISO) is used to successfully visualise hypoxia. FMISO is a cell permeable compound, which is rapidly reoxidised and moved out from the cell under normal oxygenation conditions. In hypoxic cells, FMISO is covalently bound to intracellular macromolecules and remains in the cells (Mateo et al. 2014). [18F]EF5 (2-(2-Nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide) is a lipophilic compound and has better pharmacokinetic properties than FMISO. Atherosclerotic lesions containing hypoxia have been successfully visualised with [18F] EF5 in atherosclerotic mouse models (Silvola et al. 2011). Formation of atherosclerotic plaques leads to local hypoxia. Hypoxia is able to induce increased expression of integrin αvβ3, which is related to angiogenesis (Figure 5). Elevated integrin αvβ3 expression is linked to macrophages involved in atherosclerotic inflammation (Antonov et al. 2004). Experiments with radiolabelled tri-peptide sequence arginine-glycine-asparagine based tracers such as [68Ga]NOTA-RGD and [18F]galactoRGD have increased tracer accumulation in atherosclerotic plaques (Paeng et al. 2013, Laitinen et al. 2009, Beer et al. 2014 and Haukkala et al. 2009). Also, apoptosis plays a central role in atherosclerotic inflammation and has shown to be a possible target for molecular imaging. For instance, apoptosis-specific Annexin 5 is used to visualise vascular inflammation (Laufer et al. 2008). Microcalcification is related to atherosclerotic plaque development. [18F]Sodium fluoride ([18F]NaF) is an option for identifying plaques (Figure 5) (Joshi et al. 2014). Blood monocyte Mannose receptor TSPO receptor Arterial lumen Fibrous cap Cell membrane Lipid core Adhesion molecule Macrophage Foam cell Somatostatin Glucose receptor Folate uptake receptor Calcification Neoangiogenesis Hypoxia Figure 5. Possible PET imaging targets related to atherosclerosis are macrophage infiltration, hypoxia, neoangiogenesis and microcalcification. 30 Review of the Literature 2.5.3 Left ventricular function and structure Echocardiography is one of the most important techniques for the detection and assessment of myocardial ischaemia and infarction. Cardiac ultrasound is used in the detection of the effects of myocardial ischaemia or infarction on LV function. LV size, wall thickness and myocardial wall motion can be examined by conventional 2D imaging. Diastolic and systolic volumes and EF of LV can be calculated using well-established methods. Echocardiography is a valuable tool for diagnosing MI with assessment of wall motion. MI is, in most cases, associated with regional LV motion abnormality (Greupner et al. 2012). CT is a technique that allows for the evaluation of cardiac structure and function. When combined with ECG gating and a contrast agent, high quality images can be obtained. Diastolic and systolic volumes and EF of LV can be calculated using semi-automated analysis software. LV size, wall thickness and myocardial wall motion can also be examined easily. The advances of CT imaging include high spatial resolution (Greupner et al. 2012). Cardiovascular magnetic resonance imaging (CMR) is the gold standard for the assessment of LV function (Pennell et al. 2004). The assessment of cardiac function can be implemented using CineMRI, which is accurate in the measurement of diastolic and systolic volumes and EF as well as cardiac mass (Scholtz et al. 2014). 2.5.4 Myocardial perfusion Myocardial perfusion imaging (MPI) can be performed with several techniques. Nuclear cardiology has been growing in the diagnosis and risk assessment of patients with suspected heart disease. Single-photon emission computed tomography (SPECT) was used in MPI since the early 1980s with thallium-201 (201Tl) and Technetium-99m hexakis(2-methoxy-2-methylpropylisonitrile) ([99mTc]sestamibi) after approval by the United States Food and Drug Administration (FDA) in 1990. In addition to 201Tl and [99mTc]sestamibi, a newer widely used SPECT tracer [99mTc]tetrofosmin allows evaluation of myocardial perfusion (Kelly et al. 1993). PET is increasingly used in MPI. Kinetic properties of PET tracers enable more accurate quantification of myocardial blood flow. For example, [15O]water is ideal for the blood flow quantitation (Knuuti et al. 2009). Cardiac MRI is becoming more commonly used in MPI with a gadolinium-based contrast agent. First-pass perfusion can be detected. The gradient echo pulse sequence is most commonly used to visualise perfusion. Stress perfusion MRI has a high diagnostic accuracy in detecting coronary artery disease (Scholtz et al. 2014). CT imaging allows also MPI with dynamic acquisition. Iodinated contrast bolus behaves similar to gadolinium-based contrast agents used in MRI. The most recent techniques Review of the Literature31 with 320-row CT enable MPI covering whole heart with relatively high temporal resolution (George et al. 2012). Myocardial perfusion can be assessed with echocardiography using gas-filled microbubbles, nano- or microparticles as a contrast media (Seol et al. 2014). Resting myocardial perfusion remains normal until occlusion of a coronary artery exceeds 90% (Gould et al. 1974). Stress imaging can be used to confirm perfusion defect in unclear case. Pharmacologic stress testing is used with coronary vasodilators, e.g., adenosine or regadenoson. Coronary blood flow will be increased three- to five-fold. Combining rest- and stress-imaging enables calculation of myocardial flow reserve (MFR). Resting myocardial blood flow (MBF) values can be corrected with rate-pressure product (RPP). Changes in hemodynamics during vasodilatator infusion are possible at least with a high dose of vasodilators used in experimental study settings. To maintain blood pressure, the α1adrenoceptor agonist phenylephrine can be used to oppose the systemic effects of adenosine, while leaving adenosine-induced coronary vasodilation unperturbed (Sorop et al. 2008). MPI results are usually presented using 17-segmental standardised myocardial segmentation and nomenclature for the tomographic imaging of the heart as proposed by the American Heart Association (Cerqueira et al. 2002). 2.5.5 Myocardial infarction and viability [99mTc]Sestamibi is the most used SPECT tracer for assessment of myocardial viability. Retention in myocardium is dependent on the intensity of the Na+/K+ pump in the cell membrane and reflects the intracellular level of potassium. [99mTc]Sestamibi is taken up by mitochondria (Allman 2013). [18F]FDG is a widely used PET tracer for assessing myocardial viability. PET has higher spatial resolution than SPECT. Metabolic clamping can be used for standardising myocardial glucose utilisation. The myocardium usually uses free fatty acids (FFAs) for energy production. Glucose and insulin administration before tracer injection switches energy metabolism to use more glucose than FFAs. [18F]FDG is trapped inside the viable myocyte through phosphorylation by hexokinase. The clearance from blood circulation is fast allowing high myocardium-to-blood ratios. [18F]FDG viability imaging is often combined with MPI using [13N]ammonia, [82Rb]Cl or [15O]water. Previous approaches can be replaced by a dynamic imaging with carbon-11 labelled acetate ([11C]acetate). Quantification of regional MBF and oxidative metabolism are possible with a single study (Wolpers et al. 1997). [15O]Water enables calculation of perfusable tissue fraction (PTF) and perfusable tissue index (PTI). Assessment of viability has also demonstrated to be possible by using PTF calculated by a [15O]water study (Iida et al. 2012). 32 Review of the Literature Cardiac MRI enables viability assessment by using a gadolinium-based late enhancement method to define the existence of the MI scar. The transmural extent of the MI scar is possible to visualise very accurately from MRI images with high spatial resolution (Kim et al. 2000). Viable myocardium can be estimated by measuring the contractile reserve during stress. Based on this, echocardiography can be used for the evaluation of viable myocardium (Bisplinghoff et al. 2014). 2.5.6 PET imaging of myocardial metabolism Myocardial energy expenditure can be measured with PET. FFAs are a major myocardial energy substrate under normal conditions. Energy metabolism in failing myocytes is altered and shifted to use more glucose. Myocardial glucose utilisation can be assessed with [18F]FDG or [11C]glucose and fatty acid utilisation with [18F]FTHA (14(R,S)-[18F] fluoro-6-thia-heptadecanoic acid) or [11C]palmitate (Tuunanen et al. 2011). Oxidative metabolism is affected rapidly after MI. Myocardial oxygen consumption can be estimated with [11C]acetate imaging. Myocardial efficiency, which is defined as the ratio between cardiac work and myocardial oxygen consumption, is often decreased in HF and can be measured non-invasively by combining measurement of myocardial oxygen consumption and LV work (Bengel et al. 2000). New specific radioligands make it possible to evaluate cellular changes related to postMI remodelling. For example the development of new blood vessels can be assessed with RGD–based tracers targeting αvβ3 integrin in relation with neoangiogenesis (Kiugel et al. 2014). 2.6 Positive inotropic therapy in heart failure The calcium ion (Ca2+) has a central role in myocyte contraction. The calcium ion makes the contraction possible by binding to troponin C, which leads to conformational changes in troponin I. During relaxation, Ca2+ will be released from troponin C and will be transported to sarcoplasmic reticulum or outside the cell (Figure 6). Phospholamban regulates the storing of calcium ions into the sarcoplasmic reticulum. The Na+/Ca2+ exchanger delivers calcium ions out of the cell, which is also regulated by Na+/K+ATPase. Cyclic AMP increases the activity of protein kinase A (PKA), which leads to phosphorylation of the Ca2+ channel and increased calcium ion influx into the cell. Intracellular concentration of Ca2+ increases leading to contraction. Activation of β-receptor leads to increased cyclic AMP and the activation of PKA. (Francis et al. 2014) The PKA-dependent contractility of myocardium can be increased with β-receptor agonists like dobutamine and dopamine or phosphodiesterase inhibitor, e.g., milrinone. Review of the Literature33 The Na+/Ca2+ exchanger can be regulated with cardiac glycosides (digoxin). Calcium sensitisers act by binding to troponins. Levosimendan is a calcium sensitiser used for inotropic support in acutely decompensated congestive HF. Levosimendan binds to cardiac troponin C and with the presence of calcium ions stabilises the troponin conformation. Levosimendan has also a vasodilatory effect by opening ATP sensitive potassium channels leading to vascular smooth muscle relaxation. Unlike inotropic agents in general, levosimendan has neutral effects on myocardial efficiency (Ukkonen et al. 2000). Inotropic and vasodilatory effects result in an increased contractility and decreased preload and afterload. Also, the mitochondrial ATP sensitive K+-channel-mediated cardioprotective effect is linked to levosimendan (Papp et al. 2012). Ca 2+ Cardiac glycosides + Ca cAMP AMP ATP Phosphodiesterase inhibitors β-receptor agonists 2K 3 Na PKA + Phospholamban AC Gs β Calcium sensitisers 2+ Na Sarcoplasmic reticulum Troponin complex + Troponin C Figure 6. Mechanisms of cardiomyocyte calcium metabolism and contraction. Levosimendan is a calcium sensitiser acting through binding to cardiac troponin C and with the presence of calcium ions stabilises troponin conformation. 34 3. Aims of the Study AIMS OF THE STUDY The purpose of this study was to develop and validate experimental large animal models for the use in studying coronary artery disease, MI and HF using multimodality imaging approaches. Additionally, potential radiopharmaceuticals and therapies were tested. The specific aims of the study were: 1. To investigate the feasibility of [18F]FDG PET imaging of inflammation in early coronary atherosclerotic lesions in a pig model of diabetes and hypercholesterolemia 2. To characterise cardiac remodelling in a new pig model of chronic heart failure by assessment of left ventricular functional, metabolic and structural changes using PET, CT and echocardiography 3. To characterise a bottleneck stent model for chronic myocardial ischaemia and infarction in pigs using PET imaging of myocardial perfusion 4. To test new 68Ga-labeled tracers for PET imaging of myocardial perfusion in pigs 5. To study the effects of long-term levosimendan therapy on myocardial infarct size and left ventricle function after acute coronary occlusion in a pig model of postinfarction heart failure 4. Materials and Methods35 MATERIALS AND METHODS Finnish landrace pigs were used in the experiments. All animal experiments were conducted in accordance with the European Union Directive for the use of experimental animals and approved by the national Animal Experiment Board of Finland (ELLA) and the Regional State Administrative Agency for Southern Finland (ESAVI). 4.1 Coronary artery disease model (I) 4.1.1 Experimental protocol The formation of atherosclerotic plaques was accelerated by inducing diabetes. Diabetes was induced by destroying beta cells using streptozotocin injections. Pigs were sedated with ketamine (30 mg/kg intramuscularly (i.m.), Ketalar, Amgen Technology Ireland, Dublin, Ireland) and streptozotocin 50 mg/kg (Zanosar, Pharmacia & Upjohn, Kalamazoo, MI, USA) was injected via ear vein once a day for 3 days. In order to offset insulin release from pancreas, glucose was given 25 g per os twice daily for 2 days. An atherogenic diet containing either 1.5% cholesterol and 15% lard (HF, n=6) or 4% cholesterol, 20% lard and 1.5% sodium cholate (HF+c, n=4) (Special Diet Services, Witham, UK) was started five days after the last streptozotocin injection and was continued for 6 months. Blood glucose and cholesterol levels were monitored regularly. 4.1.2 In vivo PET imaging of atherosclerotic inflammation After six months of diet intervention, [18F]FDG uptake of coronary arteries was studied by PET. Myocardial glucose uptake was suppressed with an overnight fasting and by giving a carbohydrate free diet for two days before the PET study. PET studies were performed by a PET and a 64 slice CT hybrid scanner (Discovery VCT, General Electric Medical Systems, Milwaukee, WI, USA) operated in 3-dimensional mode. List-mode acquisition for 30 minutes started 120 minutes after intravenous (i.v.) [18F]FDG injection. The acquired list-mode data was divided into cardiac and respiratory gates and were combined as dual gates compensating for both respiratory and cardiac motion. Results were presented as target-to-background ratios (TBR) calculated by dividing maximum standardised uptake value (SUV) of each coronary segment by mean SUV value of the blood. 4.1.3 Ex vivo studies After the PET study, animals were sacrificed by i.v. injection of potassium chloride (B. Braun Medical Oy, Helsinki, Finland). Coronary arteries were prepared and proximal samples were collected. Samples were weighed and [18F]FDG uptake was measured with a gamma counter (1480 Wizard 3″; PerkinElmer/Wallac, Turku, Finland). SUVs and further vessel-to-blood ratios were calculated. 36 Materials and Methods The coronary artery samples were mounted, frozen in isopentane mixed with dry ice and cut into longitudinally sections. An autoradiography study with a phosphor imaging plate (BAS-TR2025, Fuji Photo Film Co. Ltd., Tokyo, Japan) was performed. Radioactivity distribution of the 40 μm cryosections was analysed using Fluorescent Image Analyser (Fujifilm FLA-5100, Fuji Photo Film Co. Ltd., Tokyo, Japan). Cryosections of 8 μm were stained with hematoxylin & eosin (HE) and Movat’s pentachrome staining. Movat’s pentachrome stained samples were graded using a light microscope with the following scores: 1=healthy normal vessel wall, 2=intimal thickening, and 3=atheroma. Tissue sections stained with HE were digitally photographed using a light microscope. Autoradiographs and HE images were co-registered and [18F]FDG accumulation of atherosclerotic lesions and non-atherosclerotic vessel wall as photostimulated luminescence per square millimetre (PSL/mm2). Lesion-to-normal vessel wall ratios were calculated for each segment. 4.2 Surgical and percutaneous myocardial infarction model (II, III) 4.2.1 Experimental protocol Before operations or imaging studies, animals were anaesthetised by i.m. administration of midazolam 1 mg/kg (Midazolam Hameln, Hameln Pharmaceuticals GmbH, Hameln, Germany) and xylazine 4 mg/kg (Rompun vet, Bayer Animal Health GmbH, Leverkusen, Germany) (II) or with atropine (0.05 mg/kg; Leiras, Helsinki, Finland) and azaperone (Stresnil, 8 mg/kg; Janssen, Titusville, NJ, USA) (III). Animals were intubated and ventilated mechanically. Anaesthesia was maintained with i.v. infusion of propofol 10−50 mg/kg/h (Propofol Lipuro, B. Braun Melsungen AG, Melsungen, Germany) combined with fentanyl 4−10 µg/kg/h (Fentanyl-Hameln, Hameln Pharmaceuticals GmbH, Hameln, Germany). In order to induce myocardial ischaemia and infarction surgically (II), a short left anterior thoracotomy was performed. The pericardium was opened and a complete ligation of the distal LAD was made immediately after the second diagonal branch using a 5-0 monofilament polypropylene suture (Prolene, Ethicon, Norderstedt, Germany). After 15 minutes, the proximal LAD was prepared free and an ameroid constrictor (2.50 mm or 2.75 mm, model MRI-2.50-TI and MRI-2.75-TI; Research Instruments SW, Escondido, CA, USA) was placed around the LAD. Arrhythmias were prevented by administration of amiodarone (Cordarone, Sanofi-Synthelabo Ltd, Newcastle upon Tyne, UK) 8 mg/kg perorally (p.o.) daily for 1 week before and for 2 weeks after the operation. Amiodarone 6 mg/kg i.v., metoprolol 0.2 mg/kg i.v. (Seloken, Genexi, Fontenay sous Bois, France) and magnesium sulphate (MgSO4) 25 mg/kg i.v. (Addex-magnesium sulfate, Fresenius Kabi AB, Uppsala, Sweden) were administered intraoperatively. Clopidogrel 3 mg/kg p.o. (Plavix, Sanofi Winthrop Industrie S.A., Ambarès et Lagrave, France) was administered Materials and Methods37 daily 1 day before and daily for 2 weeks after the surgery to prevent premature thrombosis of the LAD. A percutaneously placed intracoronary bottleneck stent was used to induce myocardial ischaemia and infarction (III). Catheterisation was done using a GE Innova 3100IQ threedimensional (3-D) angiography device (GE Healthcare, Waukesha, WI, USA). The bottleneck stent consisted of a bare metal stent and polytetrafluoroethylene heat shrink tube. The other end of the tube was shaped into a bottleneck to restrict coronary blood flow. The construct was placed into LAD with 6-F AR-2 guiding catheter. In order to prevent arrhythmias, per oral amiodarone (Cordarone, 200 mg/day, Sanofi-Aventis, Paris, France) and bisoprolol (Bisoprolol-ratiopharm, 2.5 mg/day, Ratiopharm, Ulm, Germany) was started 1 week before stenting. Prior to stenting, 100 mg intravenous lidocaine (10 mg/ml, Orion Pharma, Espoo, Finland) and 2.5 ml MgSO4 (Addex-magnesium sulfate, 246 mg/ml, Fresenius Kabi, Uppsala, Sweden) was administered. One day before stenting, per oral acetylsalicylic acid (ASA-ratiopharm, 300 mg, Lannacher Heilmittel GmbH, Lannach, Austria) and clopidogrel (Clopidogrel Mylan, 300 mg, Mylan, Saint Priest, France) was given. ASA (100 mg/day p.o.), clopidogrel (75 mg/day p.o.) and enoxaparin (30 mg/day s.c.) were continued for one week to keep the stent open. 4.2.2 PET imaging of myocardial perfusion and viability Myocardial perfusion was measured at 3 months after the ameroid constrictor placement (II) and one week and five weeks after the bottleneck stenting (III) with [15O]water PET. MPI was done at rest and under pharmacological stress with adenosine 200 μg/ kg/min (Adenosin Life Medical, Life Medical Sweden AB, Stocksund, Sweden). The acquisition protocol consisted of following frames: 14 × 5 s, 3 × 10 s, 3 × 20 s, 4 × 30 s (total duration 4 min 40 s). The segmental LV myocardial blood flow was quantified using a single-compartment model (Iida et al. 1992). Infarcted and ischaemic myocardial regions were defined using 70% of maximum as a threshold in [15O]water PET flow quantitation at rest and during stress, respectively (III). Myocardial viability was assessed at five weeks after the bottleneck stenting with [18F] FDG PET (III). Myocardial glucose utilisation was normalised with an intravenous injection of 1 g/kg glucose and 10 IU of insulin prior tracer injection. A static 15-min PET scan was performed 40 minutes after the injection. Viability was determined as viable, partially viable or nonviable (relative [18F]FDG uptake 85, 67–85 and 67%, respectively). 4.2.3 Left ventricular size and function (II) Transthoracic echocardiography was performed for measurement of cardiac output by pulsed-wave Doppler from the LV outflow tract (LVOT) and calculated as velocity time integral × heart rate. 38 Materials and Methods End-diastolic and end-systolic volumes (EDV, ESV) and EF as well as LV mass were evaluated by contrast-enhanced CT. 4.2.4 Myocardial oxidative metabolism and efficiency (II) Myocardial perfusion and oxidative metabolism was assessed by [11C]acetate PET imaging. The scanning frames were as follows: 10 × 10 s, 1 × 60 s, 5 × 100 s, 5 × 120 s, 5 × 240 s (total duration 41 min). Myocardial blood flow was determined by the initial uptake rate (k1) of [11C]acetate (van den Hoff et al. 2001). The clearance rate constant of [11C]acetate (Kmono) was assessed reflecting myocardial oxygen consumption (Armbrecht et al. 1990). Myocardial infarct size was determined using a reduction of 40% as a cut-off for the k1 of [11C]acetate PET. Global myocardial efficiency was estimated by a work-metabolic index (WMI) with the following equation: WMI = (cardiac output) × (systolic blood pressure) / Kmono × LV mass (Ukkonen et al. 2009). Regional efficiency of the remote non-infarcted myocardium, Kmono, was studied in relation to the systolic wall stress using the following equation: Efficiency = wall stress / regional Kmono. 4.2.5 Tissue samples and histology Immediately after the imaging studies, the animals were sacrificed and heart was prepared and sliced horizontally to four slices. After 15-min incubation in 1% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, Saint Louis, MO, USA), samples were photographed and the size of infarcted region was visually estimated. Samples for histology were collected from the infarcted, non-infarcted remote (inferolateral wall). Samples were mounted and frozen in isopentane mixed with dry ice. Cryosections of 7 μm were stained with Masson’s trichrome staining. The content of fibrosis was quantified from remote sections with the colour deconvulation method using ImageJ software (Ruifrok et al. 2001). 4.3 New myocardial perfusion tracers (IV) 4.3.1 Study design The suitability of four previously discovered 68Ga-labelled ligands for MPI was tested. Results obtained with 68Ga-tracers were compared with [15O]water PET flow quantitation. Hexadentate bis(salicylaldimine) ligands, tris(3-methoxysalicylaldimine) (Tracer-1) and tris(3-ethoxysalicylaldimine) (Tracer-2) of bis(2,2-dimethyl-3aminopropyl)-ethylenediamine (BAPEN) and the bis(salicylaldimine) (Tracer-3) and Materials and Methods39 bis(3-methoxysalicylaldimine) (Tracer-4) of bis(3-aminopropyl)-dimethylenediamine (BAPDMEN) were tested in healthy pigs. 4.3.2 PET imaging and kinetic modelling of [68Ga] ligands Myocardial blood flow (MBF) was measured first with [15O]water. Then 68Ga-tracer was injected following 92-min scanning with following frames: 18 × 10 s, 4 × 30 s, 2 × 120 s, 1 × 180 s, 4 × 300 s, 6 × 600 s. For analysis of MBF with 68Ga-chelates, singlecompartment model and multiple-time graphical analyses for irreversible tracer uptake (Patlak plot) and reversible tissue uptake (Logan plot) were applied. Linear correlation between MBF measured with [15O]water and modelling results of 68Ga-ligands was calculated as using Pearson correlation. 4.3.3 Organ distribution Organ samples were collected immediately after the PET imaging. Samples of whole blood, plasma, urine, heart, lung, liver, spleen, kidney, muscle, brain, bone, bone marrow, salivary gland and abdominal fat were prepared, weighed and measured for radioactivity using a gamma counter (Wizard). Additionally, myocardium-to-liver, myocardium-tolung and myocardium-to-blood ratios were calculated. 4.3.4 In vitro binding to serum proteins Binding of 68Ga-ligands to serum proteins was determined using serum obtained from human, pig or rat. The assay was performed with the ultrafiltration method as described earlier by Basken et al. (Basken et al. 2008) and results were expressed as an unbound fraction (%). 4.4 Chronic levosimendan therapy for heart failure (V) 4.4.1 Study design The pigs had a two-step occlusion of the LAD with distal ligation and proximal ameroid constrictor. Three weeks after the surgical operation, transthoracic echocardiography was done to visualise LV wall motion (Figure 7). Clear signs of large motion abnormality in the LAD region was defined to be inclusion criteria. Animals with LV wall motion defect were allocated into control group (n=18) or levosimendan group (n=7). Levosimendan was given per orally 5 mg/kg once a day (Orion Pharma Ltd, Espoo, Finland). Intervention was continued 8 weeks and was stopped one week before terminal imaging studies. Imaging studies consisted of echocardiography, PET and CT. After imaging studies, animals were sacrificed and cardiac tissue samples were collected. 40 Materials and Methods Ligation + ameroid constrictor Echocardiography & randomisation Intervention ends one week before imaging studies 3 weeks 8 weeks Ligation + ameroid Imaging studies Intervention Imaging Blood sampling 12 weeks Blood sampling Figure 7. Study outline of the 8-week levosimendan intervention study where distal left anterior descending coronary artery (LAD) was ligated and ameroid constrictor was placed in the proximal part of LAD. 4.4.2 Effects of chronic levosimendan therapy on MI size, LV function and remodelling Myocardial infarct size was determined by TTC-stained tissue samples and using a reduction of 40% as a cut-off in the initial uptake rate (k1) of [11C]acetate PET. Left ventricular function was evaluated by measurement of end-diastolic and endsystolic volumes and ejection fraction from CT images. Cardiac remodelling was evaluated by calculation of work-metabolic index reflecting myocardial efficiency. Myocyte hypertrophy and amount of fibrosis was estimated by histology. 4.5 Statistical analyses Results were expressed as means ± standard deviation. A Shapiro-Wilk test was applied to determine whether the data were normally distributed. Correlation between study characteristics was analysed using either Pearson or Spearman correlation. Statistical significances between two study groups were evaluated by Mann-Whitney U test or two-tailed t-test. Statistical significances between more than two study groups were evaluated with one-way ANOVA and Bonferroni post hoc tests. P-values <0.05 were considered significant. 5. Results41 RESULTS 5.1 Atherosclerotic plaque inflammation (I) The principal study consisted of 10 pigs with a 6-month diet intervention. Pigs were hyperglycemic and hypercholesterlomic. Prior to imaging studies, the blood glucose level was 12.3±4.7 mmol/L and plasma total cholesterol level was 12.7±5.1 mmol/L. 5.1.1 Ex vivo studies In total, 33 coronary artery segments were prepared and studied. As histological grading based on Movat pentachrome staining, seven segments were defined as a healthy vessel wall, 16 as intimal thickening and 10 as an atheroma. Intimal thickening and atheroma lesions contained a high density of inflammatory cells. Increased [18F]FDG uptake was seen by autoradiography in coronary segments with intimal thickening and atheroma. Lesion-to-normal vessel wall ratio was 1.7±0.7 times higher in the areas of intimal thickening and 4.1±2.3 in the atheroma plaques. The ex vivo biodistribution study showed increased [18F]FDG accumulation in atheroma lesions compared to healthy coronary segments. Vessel-to-blood ratios of [18F]FDG accumulation were 1.3±0.7, 2.0±1.0 and 2.6±1.2 in the segments with no plaque, intimal thickening or atheroma lesions, respectively. 5.1.2 In vivo PET imaging Coronary [18F]FDG accumulation was visualised by dual-gated cardiac PET images coregistered with CTA image (Figure 8). [18F]FDG uptake in the myocardium was low. Average TBR was 1.1±0.5, 1.2±0.4 and 1.6±0.6 in the segments with no plaque, intimal thickening and fibroatheroma, respectively. In dual-gated PET, the highest TBR was 2.7 whereas it was only 2.0 in non-gated PET. Figure 8. Fused axial coronary CTA and dual-gated [18F]FDG PET images showing cross-section of the proximal right coronary artery (arrow) in an animal with atheroma. 42 Results 5.2 Myocardial infarction and remodelling (II, III) 5.2.1 Myocardial perfusion and viability Average MBF measured in the remote non-infarcted myocardium with [15O]water PET was comparable between pigs with distal LAD ligation and proximal ameroid constrictor and sham-operated control pigs (rest MBF: 0.97±0.32 vs. 1.24±0.40 mL/g/min, P=0.12; stress MBF: 1.61±0.75 vs. 1.94±1.00 mL/g/min, P=0.43). Also, further calculations of coronary flow reserve (CFR) and coronary vascular resistance (CVR) in the remote tissue showed no statistical differences between study groups (CFR 1.68±0.63 in ameroid pigs, 1.60±0.65 in control group, P=0.80; CVR 107.1±31.5 in ameroid pigs, 93.0±29.2 mm Hg/(mL/g/min) in control group, P=0.33) (II). In the bottleneck stented pigs, a smaller perfusion defect (24±12% of the LV) was seen at rest and larger during stress (53±10% of the LV). Four weeks after discontinuation of antiplatelet medication, large perfusion defect areas were seen in both at rest and during stress (42±13% and 54±10% of the LV, respectively) (Figure 9) (III). REST 1W STRESS 1W REST 5W REST 5W STRESS 5W STRESS 5W 0 20 40 60 80 100 % Figure 9. Inducible ischaemia seen in perfusion maps obtained with [15O]water PET at 1 week after placing a bottleneck stent into the left anterior descending coronary artery (LAD). After stopping the antiplatelet medication, the stent has occluded and caused myocardial infarction as seen at the 5-week time point. Results43 Analysis of the [18F]FDG viability study performed in bottleneck stented pigs at four weeks after discontinuation of antiplatelet medication showed that 36±3% of the LV myocardium was non-viable and 36±18% viable whereas 28±15% was defined as partially viable (III). 5.2.2 Left ventricular function (II) CT imaging showed increased LV end-diastolic and end-systolic volumes in pigs having distal ligation of the LAD and proximal ameroid constrictor compared to controls (EDV 252±84 vs. 145±17 mL, P=0.003; ESV 154±68 vs. 53±7 mL, P<0.001) (Figure 12). Ejection fraction was reduced in ameroid pigs (40±8% vs. 63±4%, P<0.001). LV mass normalised for body weight showed a higher LV mass to body weight index in ameroid pigs (1.90±0.39 vs. 1.37±0.61 g/kg, P=0.02). 5.2.3 Myocardial oxidative metabolism and efficiency (II) Analysis of the clearance rate (Kmono) of [11C]acetate showed that oxygen consumption was reduced in the infarcted regions, but was comparable in the non-infarcted remote myocardium of the ameroid pigs and sham-operated controls (remote Kmono 0.104±0.020 vs. 0.119±0.028 min-1, P=0.16). Global myocardial efficiency was lower in the ameroid than sham-operated pigs (33.2±11.1 vs. 52.7±16.6 mmHg × mL × min × g-1 × 103, P=0.005). Regional efficiency in the remote tissue was increased in ameroid pigs (995±287 vs. 647±99 mmHg × min, P=0.004). 5.2.4 Tissue samples and histology (II) TTC staining revealed that there was mostly a transmural infarct scar in the LV region vascularised by the LAD. Based on analysis of [11C]acetate data, the size of MI was 29±14% (range 14%-57%) of the LV. MI size defined by [11C]acetate perfusion and TTC staining corresponded well. Measurement of cardiomyocyte diameter in samples from the remote non-infarcted myocardium indicated increased myocyte hypertrophy in ameroid pigs (23.5±2.6 vs. 21.4±1.6 µm, P=0.04). The amount of fibrosis was comparable between the study groups (5.5±1.9% in ameroid pigs; 4.9±1.1% in control group, P=0.39). 5.3 Evaluation of new perfusion tracers (IV) 5.3.1 PET imaging and kinetic modelling of [68Ga] ligands PET imaging of four 68Ga labelled hexadentate bis(salicylaldimine) ligands showed the highest and fastest uptake into myocardium on Tracer-3 (Figure 10). 44 46 Results Results Tracer-1 Tracer-2 Tracer-3 Tracer-4 Figure 10. Representative horizontal long-axis view of heart showing the myocardial uptake of Figure 10. Representative horizontal long-axis view of heart showing the myocardial uptake of the 68 the Ga tracers at 52-92 min after the tracer injection. 68 Ga tracers at 52-92 min after the tracer injection. one-tissue compartmental model model (K1) were ModellingModelling results were Patlak plots i) and Patlak plots(K(K ) and one-tissue compartmental (K1calculated. ) were calculated. i 15 15 There was no correlation between and MBF plotted against results obtained with results [ O]water. ThereKiwas no results were MBF plotted against MBF obtained with [ O]water. 15 15 O]water. There tended to be a weak correlation between K and MBF for Tracer-1 measured with [ 1 correlation between Ki and MBF measured with [ O]water. There tended to be a weak (r=0.81, P=0.20), but not anyMBF other for tracer. and Tracer-1 (r=0.81, P=0.20), but not for any other correlation between K1for tracer. 5.3.2 Organ distribution 5.3.2 distribution labelled hexadentate bis(salicylaldimine) ligands was slightly higher in the UptakeOrgan of 68Ga myocardium in other examined organs. Accumulation in kidney andwas bile slightly were highhigher indicating Ga labelled hexadentate bis(salicylaldimine) ligands in Uptake of 68than elimination and excretion routes. Tracer-3 showed good myocardium-to-blood (7.63±1.89), the myocardium than in other examined organs. Accumulation in kidney and bile were myocardium-to-lung (3.03±0.33) and myocardium-to-liver (1.80±0.82) ratios. high indicating elimination and excretion routes. Tracer-3 showed good myocardiumto-blood (7.63±1.89), myocardium-to-lung (3.03±0.33) and myocardium-to-liver (1.80±0.82) ratios. 5.3.3 In vitro binding to serum proteins 5.3.3 In vitro binding to serum proteins A high difference in tracer binding to serum proteins was seen between human, pig and rat serum. Thehigh unbound tracer fraction was similarly between human serum pig serum A difference in tracer binding to low serum proteins was seen(9.4±5.7%) between and human, pig (5.5±2.1%), whereas was markedly higher in rat serum (45.4±20.9%). and rat serum. Theit unbound tracer fraction was similarly low between human serum (9.4±5.7%) and pig serum (5.5±2.1%), whereas it was markedly higher in rat serum (45.4±20.9%). 5.4 5.4.1 Evaluation of chronic levosimendan intervention for heart failure (V) Effects of chronic levosimendan therapy on MI size, LV function and remodelling 5.4 Evaluation of chronic levosimendan intervention for heart failure (V) 5.4.1 Effectsafter of chronic levosimendan therapy on MI size, LV function remodelling As measured the 8-week intervention period following one week washout,and myocardial infarct size measured was smallerafter in thethe levosimendan group when compared with control vs. As 8-week intervention period following oneanimals week(12±13% washout, 27±15%, P=0.03). myocardial infarct size was smaller in the levosimendan group when compared with control animals (12±13% CT imaging showed smaller vs. LV27±15%, EDV and P=0.03). ESV in the levosimendan group (EDV 161±29 mL vs. 245±84 mL, P = 0.06; ESV 81±18 mL vs. 149±67 mL, P=0.03). The ejection fraction tended to be CT imaging showed smaller LV(50±6% EDV and ESV inP=0.06). the levosimendan group (EDV 161±29 higher in the levosimendan group vs. 41±8%, mL vs. 245±84 mL, P = 0.06; ESV 81±18 mL vs. 149±67 mL, P=0.03). The ejection fraction tended to be higher in the levosimendan group (50±6% vs. 41±8%, P=0.06). Results45 Myocardial efficiency remained unchanged after intervention and was comparable between study groups (44±11 in the levosimendan group and 33±11 mmHg × mL × min × g-1 × 103 in the control group, P=0.15). Histological analysis of non-infarcted remote sections indicated that the average diameter of cardiomyocytes (levosimendan group 23.2±2.0 µm vs. control group 23.5±2.6 µm, P=0.83) and the amount of fibrosis (4.8±1.9% in the levosimendan group; 5.5±1.9% in the control group, P=0.37) were comparable between study groups. 46 6. Discussion DISCUSSION Non-invasive assessment of atherosclerotic inflammation can be beneficial to recognise patients having a high risk for cardiac events. Early detection of highly inflamed atherosclerotic lesions could give us an opportunity to identify unstable plaques, improve the care of CAD patients and reduce the risk of sudden cardiac death. Myocardial infarction often leads to adverse cardiac remodelling and finally heart failure. Oxidative metabolism and perfusion of myocardial tissue can be evaluated by [11C] acetate PET. Additionally, efficiency of cardiac work can be calculated. In this study we wanted to validate the HF model of distal LAD ligation and proximal ameroid constrictor with the evaluation of perfusion, oxidative metabolism and cardiac efficiency. As the prognosis of heart failure is poor, new therapies for the management of progression of post-MI adverse cardiac remodelling and heart failure are warranted. As earlier studies have already shown levosimendan could have multiple positive effects to prevent adverse cardiac remodelling. This study proved the beneficial effects of levosimendan as well as that the developed model of HF can be used for the evaluation of new interventions. 6.1 PET imaging of early atherosclerotic lesions Imaging of atherosclerotic inflammation by [18F]FDG PET could be valuable for identifying high-risk patients. Increased [18F]FDG accumulation exists in culprit lesions by in vivo imaging studies (Rogers et al. 2010). In this study we evaluated [18F]FDG uptake in early atherosclerotic lesions by in vivo imaging and ex vivo studies. The main finding of this study was that [18F]FDG accumulation was increased in coronary arteries with intimal thickening and atheroma as confirmed by autoradiography and histological evaluation. PET imaging of coronary arteries may be an effective method to differentiate atherosclerosis patients having highly inflamed vulnerable plaques or stable plaques with low inflammatory activity. The dual-gated method for minimising the cardiac and respiratory movement artefact increased TBR as the highest TBR in dual-gated PET was 2.7 and 2.0 in non-gated PET that is consistent with previous studies (Tarkin et al. 2014). Still, it was not statistically possible to differentiate [18F]FDG uptake between a healthy vessel wall or one with intimal thickening or an atheroma. The sensitivity of in vivo PET is probably not sensitive enough for detecting small objects with a low accumulation of imaging tracer. Advanced atherosclerotic lesions with coronary stenosis was reported in a previous study with a similar study setting that combined hyperglycemia and hypercholesterolemia (Gerrity et al. 2001 and Chatzizisis et al. 2008). However, very early atherosclerotic Discussion 47 changes were noticed in the current study. Coronary stenosis was not identified as confirmed with CTA and histology. Further studies are warranted to clarify if more advanced atherosclerotic lesions can be induced within a reasonable follow-up time. PET imaging of atherosclerotic lesions showed to be effective and the dual-gating method improved the signal-to-noise ratio. Clinically relevant CAD model could be beneficial in developing further clinical diagnostics and treatments. 6.2 Validation of a surgically induced model of myocardial infarction A translational research model to study chronic changes after MI is needed. In this study, cardiac remodelling was followed 3 months after MI. Two-step occlusion of LAD with distal ligation and proximal ameroid constrictor led to MI covering 24 percent of the LV as confirmed with [11C]acetate perfusion imaging and TTC staining of myocardial organ samples. LV size was increased over 70 percent whereas EF was decreased 37 percent. Global myocardial efficiency was decreased 37 percent, which is connected to HF. Increased LV size led to increased myocardial wall stress. Histologically-detectable myocyte hypertrophy is linked to cardiac remodelling and is a compensatory mechanism for increased wall stress. Interestingly, interstitial fibrosis was not seen as described previously in a similar study (Teramoto et al. 2011). As high as a 75 percent survival rate was reported by the authors of the original model description (Teramoto et al. 2011). In this study, the overall survival was only 26%. Still, most of unexpected sudden deaths occurred during the first week of the surgery and chronic phase survival was 44%. Sudden cardiac deaths are common in other pig studies and are probably linked to large MI size (Fallavollita et al. 2005, Huang et al. 2010 and Ishikawa et al. 2011). In this study, we confirmed the feasibility of a chronic post-MI heart failure model utilising two-step occlusion of the LAD with distal ligation followed by implantation of an ameroid constrictor in the proximal part. Left ventricular hypertrophy, impaired systolic function and LV remodelling were confirmed by imaging studies and histological evaluation. Measurements of myocardial oxidative metabolism demonstrated reduced cardiac efficiency. We conclude that this model may be used for the evaluation of new interventions. As the mortality observed in this study was very high, further development of the model of chronic HF is needed. This study furthered the understanding behind the mechanisms of cardiac remodelling. Further studies relating to ischaemic preconditioning and preventing adverse cardiac remodelling post-MI are needed. 48 Discussion 6.3 Validation of percutaneously-induced model of myocardial ischaemia and infarction Bottleneck stenting of LAD induced large ischaemic region as measured with [15O]water (24% of LV at rest and 53% of LV during stress). Discontinuing of antiplatelet medication led to an increased defect area (42% of LV at rest and 54% of LV during stress). As measured by [18F]FDG, the non-viable portion of LV was 36 percent at four weeks after discontinuing of antiplatelet medication. Infarct size was large referring to the clinical situation and this measurement makes it possible to evaluate processes after MI. An advantage of this model compared to ameroid model is that chronic myocardial ischaemia can be studied by continuing antiplatelet medication. Also, a lack of adhesion and inflammation induced by wound healing occurring after thoracic surgery noticed in surgical MI model can be beneficial in studies based on surgical interventions. Inflammation induced by wound healing can influence negatively also imaging studies of inflammation. 6.4 PET imaging and kinetic modelling of [68Ga] ligands Studied 68Ga-labelled hexadentate bis(salicylaldimine) ligands were retained in the myocardium as shown in previous study with rats (Hsiao et al. 2009). Uptake into the myocardium was slow as well as clearance from blood pool. Kinetic modelling showed that myocardial perfusion cannot be determined with these four [68Ga] ligands as compared to MBF results obtained with [15O]water. There was no significant correlation between results obtained with [15O]water and the net uptake rate Ki (Patlak plot) and K1 (one-compartmental model). One possible explanation for slow kinetics could be that tracers are very slowly moved across the cell membranes. A slow transport rate is a limiting factor for Ki and K1. An unknown factor other than perfusion may be limiting the accumulation into the myocardium. Another explanation could be a high tracer binding to serum proteins (91%-96% in pig vs. 29%-73% in rat) because the protein-bound fraction tracers are not expected to freely diffuse into tissues, which is one requirement for an effective perfusion tracer. A slow transport rate and high protein binding together could be the explanation for the lack of correlation between MBF measured with [15O]water and [68Ga] ligands as tested in healthy pigs. Despite the lack of correlation between tested [68Ga] tracers and [15O]water in this study with pigs, further studies are needed to clarify the mechanisms of the [68Ga] ligands, which showed to be promising for MPI in a previous rat study. Discussion 49 6.5 Chronic levosimendan intervention for heart failure In this study, the effects of 8-week levosimendan intervention, started three weeks after distal ligation of the LAD and placement of proximal ameroid constrictor, were studied. A significant reduction in MI size was seen in animals that received levosimendan 5 mg/kg for 8 weeks. Previously this kind of cardioprotection and reduced MI sizes were reported in a study where levosimendan therapy was initiated before inducing myocardial ischaemia and infarction (Kersten et al. 2000). In this study, a LV wall motion defect was confirmed by echocardiography before allocating animals into study groups and beginning the therapy. Animals in the levosimendan group have also smaller EDV and ESV and higher EF than animals in the control group. That is probably explained by a smaller MI size. These results are consistent with the results of previous small animal studies. Beneficial effects of levosimendan in this study are possibly explained by mitochondrial KATP channel-mediated preconditioning, which protects myocytes against ischaemia (Gross et al. 1992). Also, increased collateral blood flow has been noticed after levosimendan administration (Kersten et al. 2000). Levosimendan intervention restricted the size of MI and further cardiac remodelling, but more studies are needed to explain the exact mechanisms. Levosimendan is used for the management of acutely decompensated heart failure. Based on our results, levosimendan could have benefits also in the early treatment of acute myocardial infarction. Large animal models and clinical imaging methods used in this study enable quantitative analysis of myocardial perfusion and efficiency. This study that experimental pig model for CAD, MI, and HF could be used for further intervention studies. 6.6 Critical evaluation of the results Large animal models of atherosclerosis enable the evaluation of coronary arteries by using in vivo imaging methods. In this study we were able to induce early atherosclerotic changes including intimal thickening and atheroma-type lesions in relation to clinical coronary artery disease. This study showed how ex vivo methods can be used in a validation of in vivo results obtained by clinical imaging device. Despite the relatively long follow-up time we did not noticed any narrowing of coronary arteries with anatomical imaging methods. Also, highly inflamed coronary lesions clinically related to increased risk for cardiac events were lacking. Long follow-up led to high expenses due to costly animal housing and special high-fat diet. Also, a significant increase in body mass in short periods of time led to difficulties in animal handling. Surgical protocol consisted of distal ligation of the LAD and proximal ameroid constrictor led to large myocardial infarction following LV adverse cardiac remodelling in relation to clinical picture of myocardial infarction and heart failure. Even though we used distal 50 Discussion ligation of the LAD to induce ischaemic preconditioning, a high amount of sudden deaths were noticed in this study. Intolerance of myocardial ischaemia leading to high mortality was one important limitation in this study. Mortality in this study was higher than in HF patients and is probably not reflecting well the clinical picture. Additionally, in the clinic, HF is mainly a problem in older adults. Fairly young animals need to be used in experiments. Physiologic increases in heart mass during follow-up can be avoided by using models involving adult minipigs (Schuleri et al. 2008). A bottleneck stent placed into the LAD led to the situation mimicking clinically relevant myocardial ischaemia and infarction. Premature occlusion of coronary artery was prevented by antiplatelet medication, which can lead to bleeding complications. Also, a high quality coronary angiography device and good operator skills are needed to perform this percutaneous method. The evaluation of myocardial perfusion with 68Ga-labelled hexadentate bis(salicylaldimine) ligands was positive in rat studies. In this study with healthy pigs, these ligands were retained into myocardium, but the kinetics were very slow. We noticed that tracers were binding highly to serum proteins. Binding properties to serum proteins were very similar between pig and human (91%-96% in pig vs. 83%-96% in human), whereas it was remarkable different in rat (29%-73%). This large animal model was better for translational studies because it is more close to human metabolism than rodent models in this case. Chronic levosimendan therapy tested in chronic MI model showed to have beneficial properties to restrict the size of MI and cardiac remodelling post-MI. Limitations in this study are related to high variability in the size of MI and imbalance of study population between control and intervention group. 6.7 Future aspects Experimental pig models of atherosclerosis having myocardial ischaemia and infarction offer a versatile platform for translational research. In the future, more novel radiopharmaceuticals and interventions can be studied. Novel therapeutics can be focused on prevention of atherosclerotic plaque formation and rupture. New interventions concerning heart failure could consist of an innovative batch materials replacing infarcted myocardium. Also, gene and stem cell therapies are promising to improve cardiac function after MI. Large animal models with a human-like clinical picture are effective and valuable tools for biomedical research. Large animal models are needed because it is not possible to study complex mechanisms of atherosclerosis and HF with alternative methods like cell cultures. The size of pig enables the imaging studies of coronary arteries. In small animal models, we need to use another vessel like the aorta for modelling of atherosclerosis. In large animal studies, we can use already existing methods and devices dedicated to clinical studies and thus Discussion 51 new innovations are more easily translated into clinical use. Despite many advantages of large animal models, there are also some disadvantages, which might be taken into account in study planning like difficulties in handling of heavy animals. Large animal studies need special facilities for animal housing and operations and can lead to high expenses. The use of large animal models remains restricted to research of new concepts close to clinical application. More advanced mechanisms behind atherosclerosis and heart failure can be evaluated with multimodality imaging methods. We and others have evaluated the imaging of different stages of atherosclerotic changes with [18F]FDG PET, but it is still unsolved how to use this information in clinical imaging of atherosclerotic patients in the future. Myocardial oxidative metabolism and further assessment of cardiac efficiency is possible with [11C]acetate PET. Still, these methods are relatively rarely used in clinical imaging and thus have a huge potential in monitoring of efficacy of treatment in HF patients. Large animals enable the use of clinical imaging methods and thus new interventions can be studied in translational models before entering clinical trials. 52 7. Summary and Conclusions SUMMARY AND CONCLUSIONS On the basis of our experimental studies on atherosclerosis and heart failure, we make these conclusions: 1. Model of diabetes and hypercholesterolemia was feasible and we found an increased uptake of [18F]FDG in early coronary atherosclerotic lesions in a pig model of diabetes and hypercholesterolemia. 2. Two-step occlusion of the LAD with distal ligation and proximal ameroid constrictor resulted in a large MI and LV remodelling together with decreased myocardial efficiency during a 3-month follow-up. 3. The bottleneck stent placed in the LAD resulted in a large ischaemic region of myocardium followed by large MI after discontinuing of antiplatelet medication. 4. Evaluation of four 68Ga-labelled hexadentate bis(salicylaldimine) ligands showed no correlation between myocardial perfusion measured with [15O]water. 5. Eight-week levosimendan therapy started after recent occlusion of the LAD resulted in decreased MI size followed by attenuation of myocardial remodelling and improved systolic function. 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